WO2013148686A2 - Formulations stables d'agent de liaison à igg4 - Google Patents

Formulations stables d'agent de liaison à igg4 Download PDF

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Publication number
WO2013148686A2
WO2013148686A2 PCT/US2013/033881 US2013033881W WO2013148686A2 WO 2013148686 A2 WO2013148686 A2 WO 2013148686A2 US 2013033881 W US2013033881 W US 2013033881W WO 2013148686 A2 WO2013148686 A2 WO 2013148686A2
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WO
WIPO (PCT)
Prior art keywords
formulation
antibody
light
amino acid
cxcr5
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PCT/US2013/033881
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English (en)
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WO2013148686A3 (fr
Inventor
Julia Schnieders
Dirk Usener
Ahmed YOUSSEF
Annika Hagendorf
Martina KIRSCH
Sabrina RUGGEBERG
Original Assignee
Sanofi
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Filing date
Publication date
Priority to BR112014023952-5A priority Critical patent/BR112014023952B1/pt
Priority to ES13714505T priority patent/ES2702246T3/es
Priority to EP13714505.8A priority patent/EP2830658B1/fr
Priority to JP2015503464A priority patent/JP6265970B2/ja
Priority to EP18190556.3A priority patent/EP3431104A1/fr
Priority to BR122019026701-4A priority patent/BR122019026701B1/pt
Application filed by Sanofi filed Critical Sanofi
Priority to CA2868401A priority patent/CA2868401C/fr
Priority to RU2014142990A priority patent/RU2644214C2/ru
Priority to PL13714505T priority patent/PL2830658T3/pl
Publication of WO2013148686A2 publication Critical patent/WO2013148686A2/fr
Publication of WO2013148686A3 publication Critical patent/WO2013148686A3/fr
Priority to HK15105917.2A priority patent/HK1204990A1/xx

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the human LIGHT antigen is one potential cytokine target that has been implicated in the processes of chronic inflammatory autoimmune disease.
  • TNFSF TNF superfamily
  • LIGHT is also known as TNFSF14 or CD258. LIGHT is expressed on the surface of T cells upon activation in a tightly regulated manner. However, LIGHT is also present at detectable levels constitutively on the surface of immature dendritic cells and on T cells and natural killer (NK) cells of the gut. LIGHT mediates its biologic effects by binding three TNF superfamily receptors, including the lymphotoxin ⁇ receptor (LTpR), the herpes virus entry mediator (HVEM), and decoy receptor 3 (DcR3). LIGHT-expressing lymphocytes can induce IBD-like symptoms in humans, and increases of LIGHT expression have been observed in patients with active Crohn's disease and other inflammatory disorders such as Graft-vs.-Host Disease.
  • CXCR5 also known as Burkitt lymphoma receptor (BLR1), CD185, MDR15, and MGC117347, is a G protein-coupled receptor that is a member of the CXC chemokine receptor family.
  • the unprocessed CXCR5 precursor is 372 amino acids in length with a molecular weight of 42 K D .
  • CXCR5 has a role in B cell migration and localization within particular anatomic compartments. Knockout mice lack peripheral lymph nodes, have fewer Peyer's patches and have decreased B cell levels.
  • CXCL13 also known as BLC, is a ligand for CXCR5.
  • CXCL13 is a B cell chemoattractant.
  • Anti-LIGHT binding agents and anti-CXCR5 binding agents are each therapeutically relevant, and a need exists to formulate each of these binding agents into drug products that may be administered to subjects, particularly human subjects, for the treatment of inflammatory diseases.
  • the binding agent In order to develop a pharmaceutical formulation containing an anti- LIGHT binding agent or an anti-CXCR5 binding agent suitable for intravenous or subcutaneous administration, the binding agent must be concentrated to about 20 mg/mL or greater, usually about 100-150 mg/mL, and even up to 250 mg/mL. Many complications can arise at such high concentrations, including an increase in viscosity, a shift of pH, a change of the color of the solution, and the formation of visible and sub- visible particles.
  • binding agents are further complicated by the fact that these agents are highly prone to aggregation at such high concentrations.
  • IgG4 antibodies are even further complicated by the fact that IgG4 antibodies tend to form half- molecules at high concentrations in solution.
  • IgG4 antibodies are of therapeutic interest because they have reduced effector function.
  • Highly stable IgG4 binding agent formulations have surprisingly been found in the form of liquids and lyophilized powders that comprise an IgG4 binding agent and a citrate buffer, wherein the pH of the formulation is at or below both about pH 6 and the isoelectric point (pi) of the binding agent. These formulations improve upon conventional formulations, which often lead to dimerization of the binding agent, such as an antibody, upon increasing the concentration of the binding agent, such as an antibody, in the formulation.
  • the formulations of the invention reduce the amount of unwanted byproducts, including aggregates, half-molecules, degradation products, low molecular weight proteins (LMWPs), high molecular weight proteins (HMWPs), and rearrangements of acid, basic, and neutral isoforms of the binding agent, such as an antibody, component in the formulation.
  • unwanted byproducts including aggregates, half-molecules, degradation products, low molecular weight proteins (LMWPs), high molecular weight proteins (HMWPs), and rearrangements of acid, basic, and neutral isoforms of the binding agent, such as an antibody, component in the formulation.
  • LMWPs low molecular weight proteins
  • HMWPs high molecular weight proteins
  • rearrangements of acid, basic, and neutral isoforms of the binding agent such as an antibody, component in the formulation.
  • the invention provides a stable formulation comprising: a binding agent comprising at least a portion of a Fc region of an IgG4 antibody; and about 5 to about 50 mM citrate as a buffering agent; wherein the pH of the formulation is at or below both about pH 6 and the pi of the binding agent.
  • the binding agent is an antibody.
  • the binding agent or antibody binds to lymphotoxin-like, exhibits inducible expression and competes with herpes virus glycoprotein D for herpes virus entry mediator, a receptor expressed on lymphocytes (LIGHT).
  • the anti-LIGHT binding agent or antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of SEQ ID NOS: 1, 2, and 3, and the light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOS: 4, 5, and 6.
  • the antibody is a fully human IgG4 anti- LIGHT antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • the binding agent or antibody binds to C-X-C chemokine receptor type 5 (CXCR5).
  • CXCR5 C-X-C chemokine receptor type 5
  • the anti-CXCR5 binding agent or antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of SEQ ID NOS: 15, 16, and 17, and the light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOS: 18, 19, and 20.
  • the antibody is a humanized IgG4 anti- CXCR5 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26.
  • the antibody concentration is from about 5 to about 280 mg/mL. In certain specific embodiments of the invention, the antibody concentration is about 150 mg/mL. In other specific embodiments of the invention, the antibody concentration is about 50 mg/mL. In further specific embodiments of the invention, the antibody concentration is about 20 mg/mL. In yet further specific embodiments of the invention, the antibody concentration is about 100 mg/mL.
  • the citrate concentration is from about 5 to about 15 mM. In some embodiments of the invention, the citrate concentration is about 10 mM. In some embodiments of the invention, the citrate buffer is sodium citrate dihydrate.
  • the pH of the formulation is from about pH 5 and about pH 6. In specific embodiments of the invention, the pH of the formulation is selected from the group consisting of about pH 5.0, about pH 5.5, and about pH 6.0.
  • the pi of the binding agent or antibody is from about 6.8 and about 7.2. In alternative specific embodiments of the invention, the pi of the binding agent or antibody is from about 7.6 and about 8.4.
  • the formulation further comprises a surfactant. In certain specific embodiments of the invention, the concentration of surfactant is between about 0.001% and about 0.1% w/v. In certain embodiments of the invention, the surfactant is a polysorbate. In certain specific embodiments of the invention, the polysorbate is polysorbate 20. In some specific embodiments of the invention, the concentration of polysorbate 20 is about 0.005% w/v. In alternative specific embodiments of the invention, the concentration of polysorbate 20 is about 0.01% w/v. In further alternative specific embodiments of the invention, the concentration of polysorbate 20 is about 0.02% w/v.
  • the formulation further comprises a tonicity agent.
  • the concentration of tonicity agent is between about 0.1% and about 10% w/v.
  • the tonicity agent is a saccharide.
  • the saccharide is mannitol.
  • the concentration of mannitol is between about 1% and about 10% w/v.
  • the concentration of mannitol is about 4%.
  • the saccharide is sucrose. In some specific embodiments of the invention, the concentration of sucrose is between about 1% and about 10% w/v.
  • the concentration of sucrose is about 5% w/v. In alternative specific embodiments of the invention, the concentration of sucrose is about 6% w/v. In yet other specific embodiments of the invention, the concentration of sucrose is about 4.5% w/v.
  • the tonicity agent is sodium chloride. In some specific embodiments of the invention, the concentration of sodium chloride is between about 0.01% and about 1%. In some specific embodiments of the invention, the concentration of sodium chloride is about 0.2%. In other specific embodiments of the invention, the tonicity agent is a
  • sucrose is between about 1% and about 10% w/v. In other specific embodiments of the invention, the concentration of sucrose is between about 1% and about 10% w/v. In other specific embodiments of the invention, the concentration of sucrose is between about 1% and about 10% w/v. In other specific embodiments of the invention, the concentration of sucrose is between about 1% and about 10% w/v. In other specific embodiments of the invention, the concentration of sucrose is between about 1% and about 10% w/v. In other specific
  • the concentration of sodium chloride is between about 0.01% and about 1%. In alternative specific embodiments of the invention, the concentration of sucrose is about 6% w/v and the concentration of sodium chloride is about 0.2%. In yet further alternative specific embodiments of the invention, the concentration of sucrose is about 4.5% w/v and the concentration of sodium chloride is about 0.2%.
  • the formulation further comprises an amino acid. In certain specific embodiments of the invention, the amino acid concentration is between about 0.1% and about 5% w/v. In certain specific embodiments of the invention, the amino acid is proline or arginine. In specific embodiments of the invention, the proline or arginine concentration is between about 1% and about 2% w/v. In other specific embodiments of the invention, the proline concentration is about 1.5% w/v. In alternative specific embodiments of the invention, the arginine concentration is about 1% w/v.
  • the formulation is a liquid formulation. In other specific embodiments of the invention, the formulation is a lyophilized formulation.
  • the formulation is stable for at least 6 months at +5°C. In alterntative embodiments of the invention, the formulation is stable for at least 9 months at +5°C.
  • the formulation exhibits a reduced amount of at least one byproduct selected from the group consisting of aggregates, half-molecules, degradation products, low molecular weight proteins, high molecular weight proteins, and and rearrangements of acidic/basic/neutral isoforms of the antibody as compared to either a reference anti-LIGHT formulation comprising an anti-LIGHT antibody in phosphate buffered saline at pH 7.3 or a reference anti-CXCR5 formulation comprising an anti-LIGHT antibody in phosphate buffered saline at pH 7.3.
  • the invention provides a stable liquid antibody formulation suitable for subcutaneous administration, the formulation comprising: a) about 150 mg/mL of a fully human IgG4 anti-LIGHT (lympho toxin- like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8;
  • pH of the formulation is about pH 5.5.
  • the invention provides a stable liquid antibody formulation suitable for intravenous administration, the formulation comprising: a) about 50 mg/mL of a fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8;
  • pH of the formulation is about pH 5.5.
  • the invention provides a stable lyophilized antibody formulation suitable for intravenous administration, the formulation comprising:
  • pH of the formulation is about pH 5.5.
  • the invention provides a stable antibody formulation comprising:
  • a humanized IgG4 anti-CXCR5 (C-X-C chemokine receptor type 5) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;
  • pH of the formulation is about pH 6.0.
  • the invention provides a stable antibody formulation comprising: a) about 100 mg/mL of a humanized IgG4 anti-CXCR5 (C-X-C chemokine receptor type 5) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;
  • pH of the formulation is about pH 6.0.
  • the invention provides a kit comprising a container comprising: 1) the formulation of any one of the previous claims, and 2) a lable or instructions for the administration and use of the formulation.
  • the label comprises one or more of the following: instructions for the administration of the formulation, instructions for use of the formulation, instructions concerning the storage conditions of the formulation, information concerning lot and batch number of the formulation and/or kit, information concerning the composition of the formulation, safety information, information concerning possible adverse reactions, secondary effects, and/or side effects in connection with the administration of the formulation, or information concerning possible indications and/or contra-indications of the formulation.
  • the invention provides a pre-filled device or pre- filled container, such as a syringe, cartridge, vial, ampoule, or autoinjector comprising the formulation of the invention.
  • a kit comprising such pre-filled syringe, cartridge, vial, ampoule, or autoinjector.
  • the invention provides a method for treating an inflammatory bowel disease comprising administering to a subject in need thereof a formulation of the invention.
  • the invention provides a method for treating rheumatoid arthritis comprising administering to a subject in need thereof a formulation of the invention.
  • the invention provides a formulation for use in a method of diagnosis or treatment of the human or animal body.
  • the formulation is used in the treatment of inflammatory bowel disease.
  • the formulation is used in the treatment of rheumatoid arthritis.
  • the invention provides a method for preparing a formulation of the invention comprising mixing the components of the formulation and adjusting the pH, wherein the preparation is performed under sterile conditions or the formulation is sterilized after the mixing of the components and the pH adjustment or both.
  • the invention provides a method for preparing a stable antibody formulation comprising: a) providing an anti-LIGHT binding agent; b) resuspending the anti-LIGHT binding agent in about 5 to about 50 mM citrate buffer; and c) adjusting the pH of the formulation to about pH 5.0 to about pH 6.0.
  • Figure 1 is a picture of a gel showing the results of denatured isoelectric focusing experiments that were used to determine the isoelectric point (pi) of the fully human IgG4 anti- LIGHT antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8 formulated in phosphate buffered saline at pH 7.3 at a concentration of 5.5 mg/mL (the "Original
  • Lanes 1 & 5 IEF Calibration Kit High Range pi 5-10.5; lanes 2 & 4: a first batch of Reference Lot; lanes 3 & 4: a second batch of Reference Lot.
  • the pi values are indicated by numbers.
  • Figure 2 is a picture of an SDS-PAGE gel that compared different Reference Lot batches under reducing and non-reducing conditions.
  • Lanes 1 & 10 Biorad Precision Plus Protein Standard
  • lane 5 empty
  • lane 2 a first batch of Reference Lot under non-reduced conditions
  • lanes 3 & 4 a second batch of Reference Lot under non-reduced conditions
  • lane 6 a first batch of Reference Lot under reduced conditions
  • lanes 7 & 8 a second batch of Reference Lot under reduced conditions
  • lane 9 system control.
  • the sizes are indicated by numbers within the rows.
  • Figure 3 shows an ELISA graph that was used to determine the antigen binding activity of the first and second batches of Reference Lot.
  • FIG 4 shows a size exclusion chromatography (SEC) chromatogram of the first batch of Reference Lot.
  • SEC size exclusion chromatography
  • HMWP high molecular weight proteins
  • RRT0.8 di-/oligomers
  • LMWPs low molecular weight proteins
  • the first batch of Reference Lot batch had a purity of 97% monomer content.
  • Figure 5 shows a weak cation exchange chromatogram for the first batch of Reference Lot. As shown in Figure 5, rearrangements of acidic, neutral, and basic isoforms occured during stability studies. The first batch of Reference Lot had a distribution of acidic/neutral/basic isoforms of 42.3/55.6/1.9%.
  • Figure 6 shows a differencial scanning calorimetry thermogram of the first batch of Reference Lot. As shown in Figure 6, the three domains of the antibody unfold at 68°C, 75°C, and 78°C.
  • Figure 7 shows a dynamic light scattering pattern of the first batch of Reference Lot, which was unfiltered. DLS was used to determine the hydrodynamic diameter of the first batch of Reference Lot antibody monomer and potential soluble aggregates.
  • Figure 8 shows a dynamic light scattering pattern of the first batch of Reference Lot, which was filetered. DLS was used to determine the hydrodynamic diameter of the first batch of Reference Lot antibody monomer and potential soluble aggregates.
  • Figure 9 is a flow diagram of the drug product manufacturing process for the high antibody concentration formulation.
  • Figure 10 shows a dynamic light scattering pattern of Formulation 14. DLS was used to determine the hydrodynamic diameter of the antibody monomer and potential soluble aggregates.
  • Figure 11 is a picture of a gel showing the results of isoelectric focusing to determine the pi (isoelectric point) of the Lead CXCR5 Antibody.
  • Lanes 1,6 IEF Calibration High Range pi Kit
  • Lanes 2,4 Reference Standard Lead Antibody LP08031
  • Lanes 3,5 Lead Antibody Drug Substance, RSN0151.
  • Figure 12 is a picture of an SDS-PAGE gel that compared different drug substance batches under reducing and non-reducing conditions. The gel was also used to determine the molecular weight of the Lead CXCR5 Antibody, and the presence of any aggregates.
  • Figure 13 is an ELISA graph that was used to determine antigen binding activity of the Lead CXCR5 Antibody to a 28mer peptide of the CXCR5 antigen.
  • Figure 14 is a SEC chromatogram of stressed Lead CXCR5 Antibody.
  • SEC could detect high molecular weight proteins (HMWP), e.g., di-/oligomers or aggregates and low molecular weight proteins (LMWP) or degradation products.
  • HMWP high molecular weight proteins
  • LMWP low molecular weight proteins
  • the Lead CXCR5 Antibody had a purity of 99% monomer content.
  • Figure 15 is a WCX chromatogram that was used to determine acidic, neutral, and basic isoforms of the Lead CXCR5 Antibody.
  • the Lead CXCR5 Antibody had a distribution of acidic/neutral/basic isoforms of 14/85/1%.
  • Figure 16 is a DLS measurement that was used to determine the hydrodynamic diameter of the antibody monomer and potential soluble aggregates.
  • Figure 17 is a picture of the Lead CXCR5 Antibody in acetate buffer pH 5.0 (left) and pH 5.5 (right); each v. WFI (water for injection) and after thermal stress. This figure shows that acetate is a suitable buffer system.
  • Figure 18 is a picture of the Lead CXCR5 Antibody in histidine buffer pH 6.0 (left), pH 5.5 (middle), and pH 5.0 (right); each v. WFI (water for injection) and after thermal stress. This figure shows that histidine is a suitable buffer.
  • FIG 19 is a picture of the Lead CXCR5 Antibody in TRIS buffer pH 7.5 after UF/DF (left) and after filtration (right); each v. WFI (water for injection) and after thermal stress. This figure shows that TRIS is an incompatible buffer system.
  • Figure 20 is a picture of the Lead CXCR5 Antibody in citrate buffer pH 6.0 after UF/DF and filtration.
  • Figure 21 is a picture of the Lead CXCR5 Antibody in acetate buffer pH 5.5 after UF/DF and filtration.
  • Figure 22 is a picture of the Lead CXCR5 Antibody in succinate buffer pH 5.0 after UF/DF and filtration.
  • Figure 23 is a picture of the Lead CXCR5 Antibody in histidine buffer pH 5.0 after UF/DF and filtration.
  • Figure 24 is a picture of the Lead CXCR5 Antibody in arginine buffer pH 6.0 after UF/DF and filtration.
  • Figure 25 is a picture of the appearance of Lead CXCR5 Antibody LA_09_016 solutions with different surfactants (without surfactant, polysorbate 20, polysorbate 80, Lutrol F68, Cremophor RH40, Solutol HS15, and SDS) after mechanical stress (350 rpm, 2.5 h, RT).
  • Figure 26 is a graph that shows an increase of dimers under accelerated conditions, as analyzed by SEC. An increase of dimer formation up to 10% after three months of storage in all four histidine formulation can be seen. Acetate formulations showed an increase of dimer content up to 6%. In all four citrate formulations, the dimer concentration was below 2%, even after three months at +40°C.
  • Figure 27 is a graph showing an increase of basic isoforms under accelerated conditions, as analyzed by WCX. Histidine is worse for Lead CXCR5 Antibody stability under accelerated conditions. A slight increase of basic isoforms can be noticed for all four acetate formultions. Interestingly, it was not possible to discriminate between the four citrate formulations.
  • Figure 28 is a graph showing a decrease of neutral isoforms under accelerated conditions, as analyzed by WCX. This figure shows a strong decrease in neutral isoforms for the histidine formulations. A slight decrease was seen in acetate. Citrate was affected the least.
  • Figure 29 shows the delta pH of all four formulations (A-D) in citrate buffer at accelerated conditions.
  • the most pH stabilizing formulations are the citrate buffered, and especially formulation B and D.
  • Figure 30 shows the delta pH of all four formulations (A-D) in acetate buffer at accelerated conditions. In acetate buffered solutions of the Lead CXCR5 Antibody, the pH was shifted towards higher value.
  • Figure 31 shows the delta pH of all four formulations (A-D) in histidine buffer at accelerated conditions. In histidine buffered solutions of the Lead CXCR5 Antibody, the pH was slightly decreasing.
  • Figure 32 is a graph showing the hydrodynamic diameter of CXCR5 LA_09_027 A-D after 3 months storage at 40°C. Citrate buffered formulations showed only slight aggregates after three weeks in formulation C, and after six weeks of storage in formulation A. Some aggregates could be detected after three months in formulation B as well. But, compared to acetate buffered formulations, the amount was very little.
  • Figure 33 is a graph showing the hydrodynamic diameter of CXCR5 LA_09_028 A-D after 3 months storage at 40°C.
  • the acetate buffered formulation C showed some aggregates ⁇ 200 nm after three weeks.
  • Formulation A showed some aggregates after three months.
  • Figure 34 is a chart showing the effect of increasing Lead CXCR5 Antibody
  • FIG. 35 is a chart showing the effect of different stabilizers (excipients) on the Z- Average at 100 mg/mL of Lead CXCR5 Antibody after thermal stress. Z- Average was measured before and after thermal stress. The stabilizing effect was simiar to all tested excipients, but the increase in Z-average was generally reduced by using amino acids as stabilizers (argining, lysing, or glycine). Lysine was excluded due to a higher content of aggregates after stress. Arginine showed a better effect than glycine.
  • Figure 36 is a chart showing the effect of different stabilizers on the Z- Average at 100 mg/mL Lead CXCR5 Antibody after mechanical stress.
  • Z- Average was measured before and after mechanical stress. The same reduction in Z-average was noticed in the presence of amino acids.
  • Sucrose had a better protective effect than trehalose against mechanical stress.
  • Arginine and glycine performed better in combination with NaCl.
  • Figure 37 is a set of graphs showing particle size distribution, as measured by DLS, of Lead CXCR5 Antibody formulated in 10 mM citrate buffer at pH 6 before mechanical stress (A) and after mechanical stress (B). A higher molecular weight species was measured by DLS after mechanical stress of DS.
  • Figure 38 is a set of graphs showing particle size distribution, as measured by DLS, of Lead CXCR5 Antibody drug product prototype formulations (A-D; Table 111) before (A) and after (B) mechanical stress.
  • administer refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. , a formulation of the invention) into a patient, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • analog refers to a polypeptide that possesses a similar or identical function as a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody, but does not necessarily comprise a similar or identical amino acid sequence of a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody, or possess a similar or identical structure of a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody.
  • a polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a LIGHT or CXCR5 polypeptide (e.g., SEQ ID NO: 9 or SEQ ID NO: 14, respectively), a fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody described herein; (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a LIGHT or CXCR5 poly
  • a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoding a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody (or VH or VL region thereof) described herein.
  • a polypeptide with similar structure to a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, a LIGHT or CXCR5 epitope, or a LIGHT or CXCR5 antibody.
  • the structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallo graphic electron microscopy.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the
  • the molecules are identical at that position.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a non- limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264 2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al , 1990, J. Mol. Biol. 215:403.
  • Gapped BLAST can be utilized as described in Altschul et ah, 1997, Nucleic Acids Res. 25:3389 3402.
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • Antagonist refers to a molecule capable of inhibiting one or more biological activities of a target molecule. Antagonists may interfere with the binding of a receptor to a ligand and vice versa, by incapacitating or killing cells activated by a ligand, and/or by interfering with receptor or ligand activation ⁇ e.g., tyrosine kinase activation) or signal transduction after ligand binding to a receptor.
  • the antagonist may completely block receptor- ligand interactions or may substantially reduce such interactions. All such points of intervention by an antagonist shall be considered equivalent for purposes of the instant invention.
  • an "antagonist” or “inhibitor” of LIGHT refers to a molecule that is capable of inhibiting or otherwise decreasing one or more of the biological activities of LIGHT, such as in a cell expressing LIGHT or in a cell expressing a LIGHT ligand, such as a LIGHT receptor.
  • antibodies of the invention are antagonist antibodies that inhibit or otherwise decrease secretion of CCL20, IL-8, and/or RANTES from a cell having a cell surface-expressed LIGHT receptor ⁇ e.g., HVEM, LTpR and/or DcR3) when said antibody is contacted with said cell.
  • an antagonist of LIGHT ⁇ e.g., an antagonistic antibody of the invention
  • the anti-LIGHT antibodies are fully human, antagonistic anti-LIGHT antibodies, such as fully human, monoclonal, antagonistic anti-LIGHT antibodies.
  • an "antagonist” or “inhibitor” of CXCR5 refers to a molecule capable of inhibiting one or more biological activities, such as signaling, by CXCR5.
  • antagonists e.g., neutralizing antibodies
  • CXCR5 CXCL13 or other ligands of CXCR5, or a complex of CXCR5 and a ligand thereof, such as CXCL13
  • amino acid sequence variants or derivatives of CXCR5 or CXCL13 which antagonize the interaction between CXCR5 and a ligand, such as CXCL13
  • soluble CXCR5 optionally fused to a heterologous molecule such as an immunoglobulin region (e.g. , an immunoadhesin); a complex comprising CXCR5 in association with another receptor or biological molecule;
  • antibody immunoglobulin
  • immunoglobulin immunoglobulin
  • Ig immunoglobulin
  • the term antibody includes, but is not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g. , including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide- linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope- binding fragments of any of the above.
  • scFv single-chain Fvs
  • antibodies include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. , antigen binding domains or molecules that contain an antigen-binding site that specifically binds to a LIGHT antigen (e.g., one or more complementarity determining regions (CDRs) of an anti- LIGHT antibody) or CXCR5 antigen (e.g. , one or more complementarity determining regions (CDRs) of an anti-CXCR5 antibody).
  • LIGHT antigen e.g., one or more complementarity determining regions (CDRs) of an anti- LIGHT antibody
  • CXCR5 antigen e.g. , one or more complementarity determining regions (CDRs) of an anti-CXCR5 antibody.
  • the anti-LIGHT or anti-CXCR5 antibodies can be of any type (e.g. , IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g.
  • the anti-LIGHT antibodies are fully human, such as fully human monoclonal anti- LIGHT antibodies.
  • the anti-LIGHT antibodies are IgG antibodies, human IgG4 antibodies.
  • the anti-CXCR5 antibodies are humanized, such as humanized monoclonal anti-CXCR5 antibodies.
  • the anti-CXCR5 antibodies are IgG antibodies, humanized IgG4 antibodies.
  • anti-LIGHT antibody means an antibody or polypeptide derived therefrom (a derivative) that binds specifically to human LIGHT as defined herein, including, but not limited to, molecules that inhibit or substantially reduce the binding of LIGHT to its ligands or inhibit LIGHT activity.
  • anti-CXCR5 antibody means an antibody or polypeptide derived therefrom (a derivative) that binds specifically to human CXCR5 as defined herein, including, but not limited to, molecules that inhibit or substantially reduce the binding of CXCR5 to its ligands or inhibit CXCR5 activity.
  • B cell activity means higher than normal B cell levels, which can be local, or evidence of a biological manifestation or function of a B cell, such as antibody expression, Bruton's tyrosine kinase presence or activity, expression or presence of CD19, expression or presence of B cell activating factor and so on.
  • binding agent means any molecule, such as an antibody, a siRNA, a nucleic acid, an aptamer, a protein, or a small molecule organic compound, that binds or specifically binds to LIGHT or CXCR5, or a variant or a fragment thereof.
  • by-product includes undesired products, which detract or diminish the proportion of therapeutic/prophylactic binding agent, such as an antibody, in a given
  • typical by-products include aggregates of the antibody, fragments of the antibody, e.g. produced by degradation of the antibody by deamidation or hydrolysis, or mixtures thereof.
  • aggregates are complexes that have a molecular weight greater than the monomer antibody.
  • Antibody degradation products may include, for example, fragments of the antibody, for example, brought about by deamidation or hydrolysis.
  • degradation products are complexes that have a molecular weight less than the monomer antibody. In the case of an IgG antibody, such degradation products are less than about 150 kD.
  • composition and “formulation” are intended to encompass a product containing the specified ingredients (e.g. , an anti-LIGHT antibody or an anti-CXCR5 antibody) in, optionally, the specified amounts, as well as any product that results, directly or indirectly, from the combination of the specified ingredients in, optionally, the specified amounts.
  • specified ingredients e.g. , an anti-LIGHT antibody or an anti-CXCR5 antibody
  • constant region or “constant domain” refer to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector functions, such as interaction with the Fc receptor.
  • the terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site.
  • the constant domain contains the CHI, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.
  • CXCR5 relates to the naturally occurring, known molecule found on lymphocytes, particularly B cells, and particularly naive B cells; to such a molecule isolated from such cells; to such a molecule manufactured recombinantly using known materials and means, and using a nucleic acid encoding a CXCR5; as well as to portions of CXCR5, such as the extracellular (EC) domain, that retain the characteristics and properties relevant to the practice of the instant invention, such as CXCL13 binding.
  • a soluble CXCR5 molecule can consist essentially of the EC domain of CXCR5, which includes, generally, about the first sixty amino acids of the molecule, that is, the amino terminal portion of CXCR5.
  • CXCR5 is a non-promiscuous receptor.
  • CXCL13 is a ligand of CXCR5 and is expressed constitutively on stromal cells, such as follicular dendritic cells, and in lymphoid tissues.
  • CXCL13 specifically attracts B cells and a small subset of T cells called B helper follicular T cells, TFH. This may not be unexpected given the many interactions between T cell and B cell populations in the immune system. Moreover, activated T cells induce or upregulate CXCR5 expression. Infiltration of lymphocytes into tertiary, ectopic germinal centers (GCs) has been found to correlate well with increased disease severity and tolerance breakdown in certain disorders that present with such atypical lymph node-like structures. Using in vivo murine models, such as CXCR5-/- and CXCL13-/- mice, the absence of either the receptor or the ligand results in an altered GC fine architecture due to altered T and B cell localization, and possibly interaction.
  • GCs ectopic germinal centers
  • mice are also protected against developing severe collagen- induced arthritis (CIA).
  • CXCR5 is selectively expressed on mature B cells, which are linked to the pathogenesis of RA, blocking this receptor will modulate the arthritogenic response in affected individuals.
  • Rheumatoid arthritis treatment with biologies i.e., anti-TNFa and anti-CD20 antibodies, Rituximab
  • biologies i.e., anti-TNFa and anti-CD20 antibodies, Rituximab
  • selective targeting of CXCR5 which is only expressed on mature B cells and B helper T cells, will not affect B cell development or immunocompromise the patient.
  • the instant anti-CXCR5 antibody is a neutralizing antibody that does not mediate cell cytotoxicity.
  • CXCR5 disease is a malady, disorder, disease, condition, abnormality and so on, that is characterized by or caused by overexpression or increased levels of CXCL13 or other CXCR5 ligand, increased levels of B cells, increased levels of B cell activity, increased levels of CXCR5, or improper metabolism and activity of CXCR5.
  • epitope refers to a localized region on the surface of an antigen, such as a LIGHT or CXCR5 polypeptide, or LIGHT or CXCR5 polypeptide fragment, that is capable of being bound to one or more antigen binding regions of a binding agent, such as an antibody, and that has antigenic or immunogenic activity in an animal, such a mammal, such as in a human, that is capable of eliciting an immune response.
  • An epitope having immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal.
  • An epitope having antigenic activity is a portion of a polypeptide to which an antibody specifically binds, as determined by any method well known in the art, for example, such as an immunoassay.
  • Antigenic epitopes need not necessarily be immunogenic. Epitopes usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific three dimensional structural characteristics, as well as specific charge characteristics. A region of a polypeptide contributing to an epitope may be contiguous amino acids of the polypeptide or the epitope may come together from two or more non-contiguous regions of the polypeptide. The epitope may or may not be a three-dimensional surface feature of the antigen. In certain embodiments, a LIGHT or CXCR5 epitope is a three-dimensional surface feature of a LIGHT or CXCR5 polypeptide (e.g. , in a trimeric form of a LIGHT polypeptide).
  • a LIGHT epitope is a linear feature of a LIGHT or CXCR5 polypeptide (e.g. , in a trimeric form or monomeric form of the LIGHT polypeptide).
  • Anti- LIGHT or anti-CXCR5 antibodies may specifically bind to an epitope of the monomeric (denatured) form of LIGHT or CXCR5, an epitope of the trimeric (native) form of LIGHT or CXCR5, or both the monomeric (denatured) form and the trimeric (native) form of LIGHT or CXCR5.
  • the anti- LIGHT antibodies specifically bind to an epitope of the trimeric form of LIGHT but do not specifically bind the monomeric form of LIGHT.
  • excipients refers to inert substances that are commonly used as a diluent, vehicle, preservative, binder, stabilizing agent, etc. for drugs and includes, but is not limited to, proteins ⁇ e.g., serum albumin, etc.), amino acids ⁇ e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids ⁇ e.g., alkyl sulfonates, caprylate, etc.), surfactants ⁇ e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides ⁇ e.g., sucrose, maltose, trehalose, etc.) and polyols ⁇ e.g., mannitol, sorbitol, etc.). See, also, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in
  • fragment refers to a peptide or polypeptide that comprises less than the full length amino acid sequence. Such a fragment may arise, for example, from a truncation at the amino terminus, a truncation at the carboxy terminus, and/or an internal deletion of a residue(s) from the amino acid sequence. Fragments may, for example, result from alternative RNA splicing or from in vivo protease activity.
  • hLIGHT or hCXCR5 fragments include polypeptides comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least contiguous 100 amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a LIGHT or CXCR5 polypeptide or an antibody that specifically binds to a LIGHT or CXCR5 poly
  • Fully human antibody or “human antibody” are used interchangeably herein and refer to an antibody that comprises a human variable region and, possibly a human constant region. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin.
  • Fully human anti- LIGHT antibodies in certain embodiments, can also encompass antibodies that bind LIGHT polypeptides and are encoded by nucleic acid sequences that are naturally occurring somatic variants of a human germline immunoglobulin nucleic acid sequence. In a specific embodiment, the anti-LIGHT antibodies are fully human antibodies.
  • the term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242). Methods of producing fully human antibodies are known in the art.
  • recombinant human antibody includes human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a
  • Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, however, such recombinant human antibodies are subjected to in vitro
  • the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • an "IgG4 binding agent” or a "binding agent comprising at least a portion of an IgG4 Fc region” both refer to binding agents described herein that include at least a fragment of IgG4 Fc.
  • the fragment comprises 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or 220 amino acids of the IgG4 Fc region.
  • the fragment includes 10-50, 50-100, 100-150, or 150-200 amino acids of the IgG4 Fc region.
  • the portion of the IgG4 Fc region can have a certain homology to the IgG4 Fc region.
  • the IgG4 binding agent may include a portion of a protein with greater than 50, 60, 70, 80, 90, 93, 95, 96, 97, 98, 99, or 100% homology to the IgG4 Fc region.
  • heavy chain when used in reference to an antibody, refers to five distinct types, called alpha (a), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ), and mu ( ⁇ ), based on the amino acid sequence of the heavy chain constant domain.
  • These distinct types of heavy chains are well known in the art and give rise to five classes of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgGl, IgG3, and IgG4.
  • the heavy chain is a human heavy chain.
  • Humanized forms of non-human (e.g. , murine) antibodies are chimeric
  • immunoglobulins immunoglobulin chains or fragments thereof (such as F v , F a b, F a t,', F( a b )2 or other target-binding subsequences of antibodies) that contain sequences derived from non-human immunoglobulin, as compared to a human antibody.
  • the humanized antibody will comprise substantially all of one, and typically two, variable domains, in which all or
  • substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin template sequence.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (F c ), typically that of the human immunoglobulin template chosen.
  • F c immunoglobulin constant region
  • the goal is to have an antibody molecule that is minimally immunogenic in a human.
  • one or more amino acids in one or more CDRs also can be changed to one that is less immunogenic to a human host, without substantially minimizing the specific binding function of the one or more CDRs to CXCR5 or to CXCL13.
  • the FR can be non-human but those amino acids most immunogenic are replaced with ones less immunogenic.
  • CDR grafting is not the only way to obtain a humanized antibody. For example, modifying just the CDR regions may be insufficient as it is not uncommon for framework residues to have a role in determining the three-dimensional structure of the CDR loops and the overall affinity of the antibody for its ligand.
  • any means can be practiced so that the non-human parent antibody molecule is modified to be one that is less immunogenic to a human, and global sequence identity with a human antibody is not always a necessity.
  • humanization also can be achieved, for example, by the mere substitution of just a few residues, particularly those which are exposed on the antibody molecule and not buried within the molecule, and hence, not readily accessible to the host immune system.
  • Such a method is taught herein with respect to substituting "mobile” or “flexible” residues on the antibody molecule, the goal being to reduce or dampen the immunogenicity of the resultant molecule without comprising the specificity of the antibody for its epitope or determinant.
  • an “isolated” or “purified” binding agent such as an antibody
  • substantially free of cellular material includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • the antibody When the antibody is recombinantly produced, it is also desirable to be substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the antibody When the antibody is produced by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest.
  • anti-LIGHT or anti-CXCR5 antibodies are isolated or purified.
  • human LIGHT refers to the polypeptides ("polypeptides,” “peptides” and “proteins” are used interchangeably herein) comprising the amino acid sequence of SEQ ID NO: 9 and related polypeptides, including SNP variants thereof.
  • Related polypeptides include allelic variants ⁇ e.g., SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion
  • an anti-LIGHT binding agent such as an antibody, can bind to a LIGHT polypeptide, polypeptide fragment, antigen, and/or epitope, as an epitope is part of the larger antigen, which is part of the larger polypeptide fragment, which, in turn, is part of the larger polypeptide.
  • hLIGHT can exist in a trimeric (native) or monomeric (denatured) form.
  • human CXCR5 refers to the polypeptides ("polypeptides,” “peptides” and “proteins” are used interchangeably herein) comprising the amino acid sequence of SEQ ID NO: 14 and related polypeptides, including SNP variants thereof.
  • Related polypeptides include allelic variants (e.g., SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, in some embodiments, which retain CXCR5 activity and/or are sufficient to generate an anti-CXCR5 immune response.
  • an anti-CXCR5 binding agent such as an antibody, can bind to a CXCR5 polypeptide, polypeptide fragment, antigen, and/or epitope, as an epitope is part of the larger antigen, which is part of the larger polypeptide fragment, which, in turn, is part of the larger polypeptide.
  • Kabat numbering and like terms are recognized in the art and refer to a system of numbering amino acid residues that are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • the hypervariable region typically ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3.
  • the hypervariable region typically ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
  • light chain when used in reference to an antibody refers to two distinct types, called kappa ( ⁇ ) of lambda ( ⁇ ) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In some embodiments, the light chain is a human light chain.
  • a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as a formulation of the invention) to "manage” a LIGHT-mediated disease (e.g., chronic bowel disease, IBD, Crohn's disease, ulcerative colitis, or GVHD) or CXCR5-mediated disease (e.g., rheumatoid arthritis), one or more symptoms thereof, so as to prevent the progression or worsening of the disease.
  • therapies e.g., prophylactic or therapeutic agents, such as a formulation of the invention
  • a LIGHT-mediated disease e.g., chronic bowel disease, IBD, Crohn's disease, ulcerative colitis, or GVHD
  • CXCR5-mediated disease e.g., rheumatoid arthritis
  • a “monoclonal antibody” refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies, and each monoclonal antibody will typically recognize a single epitope on the antigen.
  • a “monoclonal antibody” is an antibody produced by a single hybridoma or other cell.
  • the term “monoclonal” is not limited to any particular method for making the antibody. For example, monoclonal antibodies may be made by the hybridoma method as described in Kohler et ah ; Nature, 256:495 (1975) or may be isolated from phage libraries.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • pharmaceutically acceptable excipient any inert substance that is combined with an active molecule, such as a monoclonal antibody, for preparing an agreeable or convenient dosage form.
  • pharmaceutically acceptable excipient is an excipient that is non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation comprising the monoclonal antibody.
  • prevent refers to the total or partial inhibition of the development, recurrence, onset or spread of a LIGHT-mediated or CXCR5- mediated disease and/or symptom related thereto, resulting from the administration of a therapy or combination of therapies provided herein (e.g. , a combination of prophylactic or therapeutic agents, such as a formulation of the invention).
  • a therapy or combination of therapies provided herein (e.g. , a combination of prophylactic or therapeutic agents, such as a formulation of the invention).
  • prophylactic agent refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a LIGHT-mediated or CXCR5- mediated disease and/or symptom related thereto in a subject.
  • prolactic agent refers to a formulation of the invention. In certain other embodiments, the term
  • prophylactic agent refers to an agent other than a formulation of the invention.
  • a prophylactic agent is an agent that is known to be useful to or has been or is currently being used to prevent a LIGHT-mediated or CXCR5-mediated disease and/or a symptom related thereto, or impede the onset, development, progression and/or severity of a LIGHT-mediated or CXCR5- mediated disease and/or a symptom related thereto.
  • the prophylactic agent is a fully human anti-LIGHT antibody, such as a fully human anti-LIGHT monoclonal antibody, or a humanized anti-CXCR5 antibody, such as a humanized anti-CXCR5 monoclonal antibody.
  • LIGHT antigen refers to that portion of a LIGHT polypeptide to which a binding agent, such as an antibody, specifically binds.
  • a LIGHT antigen also refers to an analog or derivative of a LIGHT polypeptide or fragment thereof to which a binding agent, such as an antibody, specifically binds.
  • a LIGHT antigen is a monomeric LIGHT antigen or a trimeric LIGHT antigen.
  • a region of a LIGHT polypeptide contributing to an epitope may be contiguous amino acids of the polypeptide, or the epitope may come together from two or more non-contiguous regions of the polypeptide.
  • the epitope may or may not be a three-dimensional surface feature of the antigen.
  • a localized region on the surface of a LIGHT antigen that is capable of eliciting an immune response is a LIGHT epitope.
  • the epitope may or may not be a three-dimensional surface feature of the antigen.
  • CXCR5 antigen refers to that portion of a CXCR5 polypeptide to which a binding agent, such as an antibody, specifically binds.
  • a CXCR5 antigen also refers to an analog or derivative of a CXCR5 polypeptide or fragment thereof to which a binding agent, such as an antibody, specifically binds.
  • a region of a CXCR5 polypeptide contributing to an epitope may be contiguous amino acids of the polypeptide, or the epitope may come together from two or more non-contiguous regions of the polypeptide.
  • the epitope may or may not be a three- dimensional surface feature of the antigen.
  • a localized region on the surface of a CXCR5 antigen that is capable of eliciting an immune response is a CXCR5 epitope.
  • the epitope may or may not be a three-dimensional surface feature of the antigen.
  • LIGHT- mediated disease and “LIGHT-mediated disorder” are used interchangeably and refer to any disease that is completely or partially caused by or is the result of LIGHT.
  • LIGHT is aberrantly (e.g. , highly) expressed on the surface of a cell.
  • LIGHT may be aberrantly upregulated on a particular cell type.
  • normal, aberrant, or excessive cell signaling is caused by binding of LIGHT to a LIGHT ligand.
  • the LIGHT ligand is a LIGHT receptor (e.g., HVEM, LTpR, or DCR3), for example, that is expressed on the surface of a cell, such as a colonic epithelial cell.
  • the LIGHT-mediated disease is a chronic bowel disease, an inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC).
  • IBD inflammatory bowel disease
  • CD Crohn's disease
  • UC ulcerative colitis
  • the LIGHT-mediated disease is graft- versus-host disease (GVHD).
  • CXCR5- mediated disease and “CXCR5- mediated disorder” are used interchangeably and refer to any disease that is completely or partially caused by or is the result of CXCR5.
  • CXCR5 is aberrantly (e.g. , highly) expressed on the surface of a cell.
  • CXCR5 may be aberrantly upregulated on a particular cell type.
  • normal, aberrant, or excessive cell signaling is caused by binding of CXCR5 to a CXCR5 ligand.
  • the CXCR5 ligand is CXCL13.
  • the CXCR5- mediated disease is rheumatoid arthritis (RA).
  • saccharides refers to a class of molecules that are derivatives of polyhydric alcohols. Saccharides are commonly referred to as carbohydrates and may contain different amounts of sugar (saccharide) units, e.g., monosaccharides, disaccharides, and polysaccharides.
  • an antibody that specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity, as determined by, e.g. , radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), BIACORE, or other assays known in the art.
  • Antibodies or variants or fragments thereof that specifically bind to an antigen may be cross-reactive with related antigens. In some embodiments, antibodies or variants or fragments thereof that specifically bind to an antigen do not cross-react with other antigens.
  • An antibody or a variant or a fragment thereof that specifically binds to a LIGHT or CXCR5 antigen can be identified, for example, by
  • BIAcore immunoassays
  • a specific or selective reaction will be at least twice background signal or noise, and more typically more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion regarding antibody specificity.
  • a “stable” or “stabilized” formulation is one in which the binding agent, such as an antibody, therein essentially retains its physical stability, identity, integrity, and/or chemical stability, identity, integrity, and/or biological activity upon storage.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993), for example. Stability can be measured at a selected temperature and other storage conditions for a selected time period.
  • the stability may be determined by at least one of the methods selected from the group consisting of visual inspection, SDS-PAGE, IEF, HPSEC, RFFIT, and kappa/lambda ELISA.
  • an antibody "retains its physical stability" in a pharmaceutical formulation, if it shows no signs of aggregation, precipitation, and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering, SDS-PAGE, or by (high pressure) size exclusion
  • HPSEC chromatography
  • 5% or less, typically 4% or less, typically 3% or less, more typically 2% or less, and particularly 1% or less of the antibodies forms aggregates, as measured by HPSEC or any other suitable method for measuring aggregation formation.
  • an antibody is considered stable in a particular formulation if the antibody monomer has a purity of about 90%, typically about 95%, in particular about 98% after a certain predetermined period of time under certain storage conditions in a particular formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Chemical alteration may involve size modification (e.g., clipping), which can be evaluated using (HP)SEC, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example.
  • size modification e.g., clipping
  • SDS-PAGE SDS-PAGE
  • MALDI/TOF MS matrix-assisted laser desorption ionization/time-of-flight mass spectrometry
  • charge alteration e.g. , occurring as a result of deamidation
  • ion-exchange chromatography for example.
  • An antibody "retains its biological activity" in a pharmaceutical formulation at a given time, if the biological activity of the antibody at a given time is at least about 90% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared, as determined in an antigen binding assay or virus neutralizing assay, for example.
  • a subject is typically a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g. , monkey and human), and in some embodiments a human.
  • the subject is a mammal, such as a human, having a LIGHT-mediated or CXCR5- mediated disease.
  • the subject is a mammal, such as a human, at risk of developing a LIGHT-mediated or CXCR5- mediated disease.
  • terapéuticaally effective amount refers to the amount of a therapy (e.g. , a formulation of the invention) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease and/or a symptom related thereto. This term also encompasses an amount necessary for the reduction or amelioration of the advancement or progression of a given disease, reduction or amelioration of the recurrence, development or onset of a given disease, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g. , a therapy other than a formulation of the invention).
  • a therapy e.g. , a formulation of the invention
  • the therapeutically effective amount of an antibody of the invention is from about 0.1 mg/kg (mg of antibody per kg weight of the subject) to about 100 mg/kg.
  • a therapeutically effective amount of an antibody provided therein is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg about 90 mg/kg or about 100 mg/kg (or a range therein).
  • therapeutically effective amount as used herein also refers to the amount of an antibody of the invention to achieve a specified result (e.g. , inhibition of a LIGHT biological activity of a cell, such as inhibition of secretion of CCL20, IL-8, or RANTES from the cell; or inhibition of a CXCR5 biological activity of a cell, such as binding to CXCL13).
  • a specified result e.g. , inhibition of a LIGHT biological activity of a cell, such as inhibition of secretion of CCL20, IL-8, or RANTES from the cell; or inhibition of a CXCR5 biological activity of a cell, such as binding to CXCL13).
  • therapeutic agent refers to any agent that can be used in the treatment, management or amelioration of a LIGHT-mediated or CXCR5-mediated disease and/or a symptom related thereto.
  • therapeutic agent refers to a formulation of the invention.
  • therapeutic agent refers to an agent other than a formulation of the invention.
  • a therapeutic agent is an agent that is known to be useful for, or has been or is currently being used for the treatment, management or amelioration of a LIGHT-mediated or CXCR5-mediated disease or one or more symptoms related thereto.
  • the term “therapy” refers to any protocol, method, and/or agent that can be used in the prevention, management, treatment, and/or amelioration of a LIGHT-mediated disease (e.g., IBD or GVHD) or CXCR5- mediated disease (e.g. , rheumatoid arthritis).
  • a LIGHT-mediated disease e.g., IBD or GVHD
  • CXCR5- mediated disease e.g. , rheumatoid arthritis
  • the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the prevention, management, treatment, and/or amelioration of a LIGHT- mediated or CXCR5- mediated disease known to one of skill in the art, such as medical personnel.
  • treat refers to the reduction or amelioration of the progression, severity, and/or duration of a LIGHT-mediated disease (e.g. , chronic bowel disease, IBD, or GVHD) or CXCR5- mediated disease (e.g., rheumatoid arthritis) resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as a formulation of the invention).
  • a LIGHT-mediated disease e.g. , chronic bowel disease, IBD, or GVHD
  • CXCR5- mediated disease e.g., rheumatoid arthritis
  • LIGHT refers to the reduction or inhibition of the binding of LIGHT to HVEM, the reduction or inhibition of the binding of LIGHT to LTpR, the reduction or inhibition of the binding of LIGHT to DcR3, the reduction or inhibition of the production or secretion of CCL20 from a cell expressing a LIGHT receptor of a subject, the reduction or inhibition of the production or secretion of IL-8 from a cell expressing a LIGHT receptor of a subject, the reduction or inhibition of the production or secretion of RANTES from a cell expressing a LIGHT receptor of a subject, and/or the inhibition or reduction of one or more symptoms associated with a LIGHT-mediated disease, such as a chronic bowel disease, IBD, or GVHD.
  • a LIGHT-mediated disease such as a chronic bowel disease, IBD, or GVHD.
  • CXCR5 refers to the reduction or inhibition of the binding of CXCR5 to CXCL13, and/or the inhibition or reduction of one or more symptoms associated with a CXCR5- mediated disease, such as rheumatoid arthritis.
  • variable region refers to a portion of the light and heavy chains, typically about the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variability in sequence is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FR).
  • CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. Numbering of amino acid positions is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, D.C.) 5 th ed. ("Kabat et al ").
  • the variable region is a human variable region.
  • the formulations of the invention have surprisingly been found in the form of liquids and lyophilized powders that comprise an IgG4 binding agent and a citrate buffer, wherein the pH of the formulation is at or below both about pH 6 and the isoelectric point (pi) of the binding agent.
  • the formulations of the invention provide significant improvements over conventional IgG4 binding agent formulations ⁇ e.g., phosphate buffered saline (PBS) formulations), which form unwanted byproducts upon increasing the concentration of the binding agent in the formulation.
  • PBS phosphate buffered saline
  • the formulations of the invention have a reduced amount of aggregates, half-molecules, degradation products, low molecular weight proteins (LMWPs), high molecular weight proteins (HMWPs), and rearrangements of acid, basic, and neutral isoforms of the binding agent in the formulations.
  • LMWPs low molecular weight proteins
  • HMWPs high molecular weight proteins
  • the formulations of the invention include an anti-LIGHT binding agent.
  • the binding agents may be any molecule, such as an antibody, a siRNA, a nucleic acid, an aptamer, a protein, or a small molecule organic compound, that binds or specifically binds to LIGHT, or a variant or a fragment thereof.
  • the binding agent is an anti- LIGHT antibody, or a variant thereof, or an antigen binding fragment thereof.
  • Anti-LIGHT antibodies specifically bind to a LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) protein, polypeptide fragment, or epitope.
  • the LIGHT molecule may be from any species.
  • the LIGHT molecule is from a human, known herein as "hLIGHT".
  • hLIGHT has the following amino acid sequence, which is identified as SEQ ID NO: 9:
  • the anti-LIGHT antibody is a humanized antibody, a fully human antibody, or a variant thereof or an antigen-binding fragment thereof.
  • anti-LIGHT antibodies prevent binding of LIGHT with its receptors and inhibit LIGHT biological activity (e.g. , the LIGHT-mediated production or secretion of CCL20, IL-8, or RANTES from cells expressing a LIGHT ligand, such as a LIGHT receptor (e.g. , HVEM, LTpR, and/or DcR3).
  • LIGHT biological activity e.g. , the LIGHT-mediated production or secretion of CCL20, IL-8, or RANTES from cells expressing a LIGHT ligand, such as a LIGHT receptor (e.g. , HVEM, LTpR, and/or DcR3).
  • the anti-LIGHT antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of any one or more of the following complementary determining regions (CDRs):
  • the anti-LIGHT antibody comprises a light chain variable region (VL) comprising the amino acid sequence of any one or more of the following
  • CDRs complementary determining regions
  • the anti-LIGHT antibody comprises a heavy chain variable region (VH) comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3.
  • VH heavy chain variable region
  • the anti-LIGHT antibody comprises a light chain variable region (VL) comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6.
  • VL light chain variable region
  • the anti-LIGHT antibody comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3; and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6.
  • the anti-LIGHT antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7:
  • VH variable region of the heavy chain
  • Positions 123-448 constant region of human IgG4 (SwissProt IGHG4_HUMAN with the two mutations S241P and L248E, according to Kabat numbering).
  • the anti-LIGHT antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 8:
  • Positions 108-214 constant region of human CK.
  • the anti-LIGHT antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • the anti-LIGHT antibody comprises a heavy chain derived from the amino acid sequence of SEQ ID NO: 10, which may be encoded by the nucleic acid sequence of SEQ ID NO: 11.
  • Nucleic acids 1-57 nucleic acids encoding the signal peptide
  • Nucleic acids 58-423 nucleic acids encoding the 124F19k2 variable region (VH)
  • Nucleic acids 424-end nucleic acids encoding the hIgG4 PE constant region
  • the anti- LIGHT antibody comprises a light chain derived from the amino acid sequence of SEQ ID NO: 12, which may be encoded by the nucleic acid sequence of SEQ ID NO: 13.
  • Amino acids 23-129 124F19k2 variable region (VL) Amino acids 130-end: hKappa constant region
  • Nucleic acids 1-66 nucleic acids encoding the signal peptide
  • Nucleic acids 67-387 nucleic acids encoding the 124F19k2 variable region (VL)
  • Nucleic acids 388-end nucleic acids encoding the hKappa constant region
  • the anti-LIGHT antibody is a fully human antibody.
  • Examples of fully human antibody isotypes include IgA, IgD, IgE, IgG, and IgM.
  • the anti-LIGHT antibody is an IgG antibody. There are four forms of IgG.
  • the anti-LIGHT antibody is an IgG4 antibody.
  • the anti-LIGHT antibody is a fully human IgG4 antibody.
  • the anti-LIGHT antibody further comprises a constant region, e.g., a human IgG constant region.
  • the constant region is a human IgG4 constant region.
  • the constant region is a modified human IgG4 constant region.
  • the constant region is a human CK constant region.
  • the anti-LIGHT antibody is a fully human IgG4 anti-LIGHT antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8 (the "Lead LIGHT Antibody”).
  • the anti-LIGHT antibody is a fully human IgG4 anti-LIGHT antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising 3 complementary determining regions (CDRs) comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and the light chain variable region comprising 3 CDRs comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6.
  • formulations of the invention also include variants of anti- LIGHT binding agents, such as antibodies.
  • the formulations of the invention may include variants of the anti-LIGHT antibody that is a fully human IgG4 anti-LIGHT antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8.
  • variants of anti-LIGHT antibodies may have similar physicochemical properties based on their high similarity, and therefore are also included within the scope of the invention.
  • Variants are defined as antibodies with an amino acid sequence that is at least 95%, at least 97%, for instance at least 98% or 99% homologous to an anti-LIGHT antibody, and capable of competing for binding to a LIGHT polypeptide, a LIGHT polypeptide fragment, or a LIGHT epitope.
  • the variants will ameliorate, neutralize, or otherwise inhibit LIGHT biological activity (e.g. , the LIGHT-mediated production or secretion of CCL20, IL-8, or RANTES from cells expressing a LIGHT ligand, such as a LIGHT receptor (e.g. , HVEM, LTpR, and/or DcR3).
  • LIGHT biological activity e.g. , the LIGHT-mediated production or secretion of CCL20, IL-8, or RANTES from cells expressing a LIGHT ligand, such as a LIGHT receptor (e.g. , HVEM, LTpR, and/or DcR3).
  • the variants are human antibodies, and, in some embodiments, are IgG4 molecules.
  • a variant is at least 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence with the Lead Antibody.
  • the term "variant" refers to an antibody that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequences of the anti-LIGHT antibody.
  • the variant may have conservative sequence modifications, including amino acid substitutions, modifications, additions, and deletions.
  • modifications include, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and linkage to a cellular ligand or other protein.
  • Amino acid modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis, molecular cloning, oligonucleotide-directed mutagenesis, and random PCR-mediated
  • Conservative amino acid substitutions include the ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g. , aspartic acid, glutamic acid), uncharged polar side chains (e.g. , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g. , aspartic acid, glutamic acid
  • uncharged polar side chains e.g. , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g
  • glycine alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g. , threonine, valine, isoleucine
  • aromatic side chains e.g. , tyrosine, phenylalanine, tryptophan.
  • Guidance in determining which amino acid residues may be substituted, modified, inserted, or deleted without abolishing immunological activity may be found using computer programs well known in the art.
  • Computer algorithms such as, inter alia, Gap or Bestfit, which are known to a person skilled in the art, can be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues.
  • Variants may have the same or different, either higher or lower, binding affinities compared to an anti-LIGHT antibody, but are still capable of specifically binding to LIGHT, and may have the same, higher or lower, biological activity as the anti-LIGHT antibody.
  • Embodiments of the invention also include antigen binding fragments of the anti-LIGHT binding agents, such as antibodies.
  • antigen binding domain refers to that portion of an antibody which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the complementary determining regions (CDR)).
  • CDR complementary determining regions
  • the antigen binding region can be derived from any animal species, such as rodents (e.g. , rabbit, rat or hamster) and humans. In some embodiments, the antigen binding region will be of human origin.
  • Non-limiting examples of antigen binding fragments include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single chain Fv (scFv) molecules, dAb fragments, and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of the antibody.
  • the anti-LIGHT binding agents (or a variant thereof or an antigen binding fragment thereof) will ameliorate, neutralize, or otherwise inhibit LIGHT biological activity in vivo (e.g. , the LIGHT- mediated production or secretion of CCL20, IL-8, or RANTES from a cell expressing a LIGHT receptor, e.g. , HVEM, LTpR, and/or DcR3).
  • LIGHT biological activity in vivo e.g. , the LIGHT- mediated production or secretion of CCL20, IL-8, or RANTES from a cell expressing a LIGHT receptor, e.g. , HVEM, LTpR, and/or DcR3
  • the anti-LIGHT binding agents are antagonist binding agents that ameliorate, neutralize, or otherwise inhibit LIGHT biological activity in vivo (e.g. , the LIGHT-mediated production or secretion of CCL20, IL-8, or RANTES from cells expressing a LIGHT ligand, such as a LIGHT receptor, (e.g., HVEM, LTpR, and/or DcR3).
  • a LIGHT ligand such as a LIGHT receptor, (e.g., HVEM, LTpR, and/or DcR3).
  • the anti-LIGHT binding agent (or a variant thereof or an antigen binding fragment thereof) is present in the formulations in an amount from about 5 mg/mL to about 280 mg/mL, e.g. , about 5 mg/mL to about 200 mg/mL, about 50 mg/mL to about 250 mg/mL, about 100 mg/mL to about 250 mg/mL, about 50 mg/mL to about 200 mg/mL, and about 100 mg/mL to about 200 mg/mL.
  • the anti- LIGHT binding agent may be present in the formulation in an amount of about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, about 100 mg/mL, about 105 mg/mL, about 110 mg/mL, about 115 mg/mL, about 120 mg/mL, about 125 mg/mL, about 130 mg/mL, about 135 mg/mL, about 140 mg/mL, about 145 mg/mL, about 150 mg/mL, about 155 mg/mL, about 160 mg/mL, about 165 mg//
  • the anti-LIGHT binding agent may be present in the formulation in an amount from about 5 to about 25 mg/mL, from about 26 to about 50 mg/mL, from about 51 to about 75 mg/mL, from about 76 to about 100 mg/mL, from about 101 to about 125 mg/mL, from about 126 to about 150 mg/mL, from about 151 to about 175 mg/mL, from about 176 to about 200 mg/mL, from about 201 mg/mL to about 225 mg/mL, from about 226 mg/mL to about 250 mg/mL, from about 251 to about 280 mg/mL, from about 5 to about 10 mg/mL, from about 40 to about 60 mg/mL, from about 75 to about 85 mg/mL, or from about 140 to about 160 mg/mL.
  • the anti-LIGHT binding agent is present in the formulation in an amount from about 50 mg/mL to about 170 mg, about 100 mg/mL to about 160 mg/mL, for example about 150 mg/mL.
  • the anti-LIGHT binding agent is present in an amount of about 50 mg/mL.
  • a fully human IgG4 anti- LIGHT antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8 is present in the formulation in an amount of about 150 mg/mL.
  • the formulations of the invention include an anti-CXCR5 binding agent.
  • the binding agents may be any molecule, such as an antibody, a siRNA, a nucleic acid, an aptamer, a protein, or a small molecule organic compound, that binds or specifically binds to CXCR5, or a variant or a fragment thereof.
  • the binding agent is an anti-CXCR5 antibody, or a variant thereof, or an antigen binding fragment thereof.
  • Anti-CXCR5 antibodies specifically bind to a CXCL13 (also known as BLC) protein, polypeptide fragment, or epitope.
  • the CXCR5 molecule may be from any species.
  • the CXCR5 molecule is from a human, known herein as "hCXCR5".
  • hCXCR5 has the following amino acid sequence, which is identified as SEQ ID NO: 14:
  • the anti-CXCR5 antibody is a humanized antibody, a fully human antibody, or a variant thereof or an antigen-binding fragment thereof.
  • anti-CXCR5 antibodies prevent binding of CXCR5 with its ligands and inhibit CXCR5 biological activity (e.g. , the binding of CXCR5 to CXCL13).
  • the anti-CXCR5 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of any one or more of the following complementary determining regions (CDRs):
  • the anti-CXCR5 antibody comprises a light chain variable region (VL) comprising the amino acid sequence of any one or more of the following complementary determining regions (CDRs):
  • LCDR1 - RSSKSLLHSSGKTYLY (SEQ ID NO: 18); LCDR2 - RLSSLA (SEQ ID NO: 19); or
  • the anti-CXCR5 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequences of SEQ ID NOs: 15, 16, and 17.
  • VH heavy chain variable region
  • the anti-CXCR5 antibody comprises a light chain variable region (VL) comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 20.
  • VL light chain variable region
  • the anti-CXCR5 antibody comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 15, 16, and 17; and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 20.
  • the anti-CXCR5 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21:
  • the anti-CXCR5 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22:
  • DIVMTQAAPS VAVTPGASVS I SCRS SKSLL HS SGKTYLYW FLQRPGQSPQ LL IYRLS SLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV YYCMQHLEYP YTFGGGTKLE IK (SEQ ID NO: 22) .
  • the anti-CXCR5 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22.
  • the anti-CXCR5 antibody further comprises a constant region, e.g., a human IgG constant region.
  • the constant region is a human IgG4 constant region.
  • the constant region is a modified human IgG4 constant region.
  • the human IgG4 constant region has the following modifications: S241P (shown below in SEQ ID NO: 23 in bold), L248E (shown below in SEQ ID NO: 23 in bold), and the lack of a terminal lysine in order to avoid heterogeneity.
  • the IgG4 constant region comprises the amino acid sequence of SEQ ID NO: 23:
  • ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQS S GLYSLS SWT VPS S SLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMI SRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS S IEKT I SKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG (SEQ ID NO: 23) .
  • the constant region is a human CK constant region.
  • the CK constant region comprises the amino acid sequence of SEQ ID NO: 24:
  • RTVAAPSVF I FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLS S TLTLSKADYE KHKVYACEVT HQGLS SPVTK SFNRGEC (SEQ ID NO: 24) .
  • the anti-CXCR5 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25:
  • VH variable region of the heavy chain
  • Positions 112-432 constant region of human IgG4 (SwissProt IGHG4_HUMAN, including the following modifications: S241P, L248E, and the lack of a terminal lysine in order to avoid heterogeneity).
  • the anti-CXCR5 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 26:
  • Positions 1-112 variable region of the light chain (VL).
  • VL variable region of the light chain
  • the CDRs complementarity determining regions, according to Kabat definition) are underlined.
  • Positions 113-182 constant region of human CK.
  • the anti-CXCR5 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a light chain comprising the amino acid sequence of SEQ ID NO: 26.
  • the anti-CXCR5 antibody further comprises a leader sequence.
  • the leader sequence in some embodiments, comprises an amino acid sequence from 1-30 amino acids in length, such as 25-25 amino acids, and typically 19 amino acids.
  • the heavy chain, light chain, or both the heavy and light chain may comprise a leader sequence.
  • the leader sequence comprises the amino acid sequence of SEQ ID NO: 16: MGWSCIILFL VATATGVHS (SEQ ID NO: 27) .
  • the anti-CXCR5 antibody comprises a heavy chain derived from the amino acid sequence of SEQ ID NO: 28:
  • MGWSCIILFL VATATGVHSQ VQLKESGPGL VAPSESLSIT CTVSGFSLID YGVNWIRQPP GKGLEWLGVI WGDGTTYYNP SLKSRLSISK DNSKSQVFLK VTSLTTDDTA MYYCARIVYW GQGTLVTVSA ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLD
  • Positions 20-130 variable region of the heavy chain (VH).
  • VH variable region of the heavy chain
  • the CDRs complementarity determining regions, according to Kabat definition) are underlined.
  • Positions 131-456 constant region of human IgG4 (SwissProt IGHG4_HUMAN, including the following modifications: S241P, L248E, and the lack of a terminal lysine in order to avoid heterogeneity).
  • the anti-CXCR5 antibody comprises a light chain derived from the amino acid sequence of SEQ ID NO: 29:
  • MGWSCIILFL VATATGVHSD IVMTQAAPSV AVTPGASVSI SCRSSKSLLH SSGKTYLYWF LQRPGQSPQL LIYRLSSLAS GVPDRFSGSG SGTAFTLRIS RVEAEDVGVY YCMQHLEYPY TFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO: 29) .
  • Positions 20-131 variable region of the light chain (VL).
  • the CDRs complementarity
  • the anti-CXCR5 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 28, and a light chain comprising the amino acid sequence of SEQ ID NO: 29.
  • the anti-CXCR5 antibody is a humanized or a fully human antibody.
  • humanized and fully human antibody isotypes include IgA, IgD, IgE, IgG, and IgM.
  • the anti-CXCR5 antibody is an IgG antibody. There are four forms of IgG.
  • the anti-CXCR5 antibody is an IgG4 antibody.
  • the anti-CXCR5 antibody is a humanized IgG4 antibody.
  • the anti-CXCR5 antibody is a humanized IgG4 anti-CXCR5 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26 (the "Lead CXCR5 Antibody").
  • the anti-CXCR5 antibody is a humanized IgG4 anti-CXCR5 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising 3 complementary determining regions (CDRs) comprising the amino acid sequences of SEQ ID NOs: 15, 16, and 17, and the light chain variable region comprising 3 CDRs comprising the amino acid sequences of SEQ ID NOs: 18, 19, and 20.
  • CDRs complementary determining regions
  • anti-CXCR5 antibodies including the anti-CXCR5 antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO: 25 and a light chain amino acid sequence comprising SEQ ID NO: 26, have been described in detail in PCT Publication WO 2009/032661, which are incorporated herein by reference.
  • formulations of the invention also include variants of anti- CXCR5 binding agents, such as antibodies.
  • the formulations of the invention may include variants of the anti-CXCR5 antibody that is a humanized IgG4 anti-CXCR5 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26.
  • Variants of anti-CXCR5 antibodies may have similar physicochemical properties based on their high similarity, and therefore are also included within the scope of the invention.
  • Variants are defined as antibodies with an amino acid sequence that is at least 95%, at least 97%, for instance at least 98% or 99% homologous to an anti-CXCR5 antibody, and capable of competing for binding to a CXCR5 polypeptide, a CXCR5 polypeptide fragment, or a CXCR5 epitope.
  • the variants will ameliorate, neutralize, or otherwise inhibit CXCR5 biological activity (e.g. , the binding of CXCL13 to CXCR5). Determining competition for binding to the target can be done by routine methods known to the skilled person in the art.
  • the variants are human antibodies, and, in some embodiments, are IgG4 molecules.
  • a variant is at least 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence with the Lead Antibody.
  • variant refers to an antibody that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequences of the anti-CXCR5 antibody.
  • the variant may have conservative sequence modifications, including amino acid substitutions, modifications, additions, and deletions.
  • modifications include, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and linkage to a cellular ligand or other protein.
  • Amino acid modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis, molecular cloning, oligonucleotide-directed mutagenesis, and random PCR-mediated
  • Conservative amino acid substitutions include the ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g. , aspartic acid, glutamic acid), uncharged polar side chains (e.g. , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g. , aspartic acid, glutamic acid
  • uncharged polar side chains e.g. , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g
  • glycine alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g. , threonine, valine, isoleucine
  • aromatic side chains e.g. , tyrosine, phenylalanine, tryptophan.
  • Guidance in determining which amino acid residues may be substituted, modified, inserted, or deleted without abolishing immunological activity may be found using computer programs well known in the art.
  • Computer algorithms such as, inter alia, Gap or Bestfit, which are known to a person skilled in the art, can be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues.
  • Variants may have the same or different, either higher or lower, binding affinities compared to an anti-CXCR5 antibody, but are still capable of specifically binding to CXCR5, and may have the same, higher or lower, biological activity as the anti-CXCR5 antibody.
  • Embodiments of the invention also include antigen binding fragments of the anti-CXCR5 binding agents, such as antibodies.
  • the term "antigen binding domain,” “antigen binding region,” “antigen binding fragment,” and similar terms refer to that portion of an antibody which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the complementary determining regions (CDR)).
  • the antigen binding region can be derived from any animal species, such as rodents (e.g. , rabbit, rat or hamster) and humans. In some embodiments, the antigen binding region will be of human origin.
  • Non-limiting examples of antigen binding fragments include: Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single chain Fv (scFv) molecules, dAb fragments, and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of the antibody.
  • the anti-CXCR5 binding agents (or a variant thereof or an antigen binding fragment thereof) will ameliorate, neutralize, or otherwise inhibit CXCR5 biological activity in vivo (e.g. , the binding of CXCL13 to CXCR5).
  • the anti-CXCR5 binding agents are antagonist binding agents that ameliorate, neutralize, or otherwise inhibit CXCR5 biological activity in vivo (e.g. , the binding of CXCL13 to CXCR5).
  • the anti-CXCR5 binding agent (or a variant thereof or an antigen binding fragment thereof) is present in the formulations in an amount from about 5 mg/mL to about 280 mg/mL, e.g., about 5 mg/mL to about 200 mg/mL, about 5 mg/mL to about 125 mg/mL, about 5 mg/mL to about 75 mg/mL, about 5 mg/mL to about 50 mg/mL, and about 5 mg/mL to about 25 mg/mL.
  • the anti-CXCR5 binding agent may be present in the formulation in an amount of about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL, about 90 mg/mL, about 95 mg/mL, about 100 mg/mL, about 105 mg/mL, about 110 mg/mL, about 115 mg/mL, about 120 mg/mL, about 125 mg/mL, about 130 mg/mL, about 135 mg/mL, about 140 mg/mL, about 145 mg/mL, about 150 mg/mL, about 155 mg/mL, about 160 mg/mL, about 165
  • the anti-CXCR5 binding agent may be present in the formulation in an amount from about 5 to about 25 mg/mL, from about 26 to about 50 mg/mL, from about 51 to about 75 mg/mL, from about 76 to about 100 mg/mL, from about 101 to about 125 mg/mL, from about 126 to about 150 mg/mL, from about 151 to about 175 mg/mL, from about 176 to about 200 mg/mL, from about 201 mg/mL to about 225 mg/mL, from about 226 mg/mL to about 250 mg/mL, from about 251 to about 280 mg/mL, from about 5 to about 25 mg/mL, from about 40 to about 60 mg/mL, from about 75 to about 85 mg/mL, or from about 90 to about 110 mg/mL.
  • the anti-CXCR5 binding agent is present in the formulation in an amount of about 20 mg/mL.
  • the anti-CXCR5 binding agent is present in an amount of about 100 mg/mL.
  • a humanized IgG4 anti-CXCR5 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26 is present in the formulation in an amount of about 20 mg/mL or 100 mg/mL.
  • the formulations of the invention comprise a citrate buffer as a buffering agent.
  • a buffering agent maintains a physiologically suitable pH.
  • a buffering agent enhances isotonicity and chemical stability of the formulation.
  • the citrate buffer is present in the formulations at a concentration from about 0.5 mM to about 50 mM, e.g., about 5 mM to about 15 mM.
  • the citrate buffer may be present in the formulation at a concentration about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM,
  • the citrate buffer is present in the formulation at a concentration from about 7 mM to about 13 mM, such as from about 9 mM to about 11 mM. In some embodiments, the citrate buffer is present at a concentration of about 10 mM.
  • the formulations of the invention have a pH at or below pH 6.
  • the pH of the formulations ranges from about 5.0 to about 6.0.
  • the pH of the formulations may be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, and about 6.0.
  • the pH of the formulations may range from about 5.5 to about 6.0.
  • the pH is either about 5.5 or about 6.0.
  • the pH of the formulation may be measured by any means known to those of skill in the art. A means for measuring pH is using a pH meter with a micro-electrode.
  • the formulations of the invention have a pH at or below the isoelectric point (pi) of the binding agent, such as an antibody.
  • the isoelectric point is the pH at which a particular molecule or surface carries no net electrical charge.
  • the pi of an anti-LIGHT or an anti-CXCR5 binding agent may be determined by any means known to those of skill in the art.
  • the pi of an anti-LIGHT or anti-CXCR5 antibody is determined by denaturated isoelectric focusing. As shown in Figure 1, the pi of a fully human IgG4 anti- LIGHT antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8 is 6.8-7.2.
  • the pi of a humanized IgG4 anti-CXCR5 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26 is 7.6-8.4.
  • the formulations of the invention may, optionally, further comprise a surfactant, which is also known as a stabilizing agent.
  • a surfactant which is also known as a stabilizing agent.
  • Surfactants/stabilizing agents are chemical compounds that interact and stabilize biological molecules and/or general pharmaceutical excipients in a formulation.
  • surfactants may be used in conjunction with lower temperature storage.
  • Surfactants generally protect the binding agent from air/solution interface induced stresses and solution/surface induced stresses, which may otherwise result in protein aggregation.
  • Surfactants may include, but are not limited to, polysorbates, glycerin, dicarboxylic acids, oxalic acid, succinic acid, fumaric acids, phthalic acids, and combinations thereof. Those skilled in the art are aware that other surfactants, e.g.
  • non- ionic or ionic detergents can be used as long as they are pharmaceutically acceptable, i.e. suitable for administration to subjects.
  • the surfactant is, in some embodiments, a polysorbate.
  • polysorbates include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.
  • the surfactant is present in the formulations in an amount from about 0.001% to about 0.1% (w/v).
  • the surfactant may be present in the formulations in an amount of about 0.001% (w/v), about 0.002% (w/v), about 0.003% (w/v), about 0.004% (w/v), about 0.005% (w/v), about 0.006% (w/v), about 0.007% (w/v), about 0.008% (w/v), about 0.009% (w/v), about 0.01% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.04% (w/v), about 0.05% (w/v), about 0.06% (w/v), about 0.07% (w/v), about 0.08% (w/v), about 0.09% (w/v), and about 0.1% (w/v).
  • the surfactant is present in the formulations from about 0.003% to about 0.05% (w/v), about 0.004% to about 0.025% (w/v), or about 0.005% to about 0.02% (w/v), e.g. about 0.005% (w/v).
  • polysorbate 20 may be present in an amount from about 0.001% to about 0.1% (w/v), about 0.002% to about 0.01% (w/v), about 0.003% to about 0.008% (w/v), and about 0.004% to about 0.006% (w/v), e.g., about 0.005% (w/v).
  • polysorbate 20 is present in an amount from about 0.001% to about 0.1% (w/v), about 0.005% to about 0.05% (w/v), and about 0.0075% to about 0.025% (w/v), e.g., about 0.01% (w/v). In further alternative embodiments, polysorbate 20 is present in an amount from about 0.001% to about 0.1% (w/v), about 0.005% to about 0.05% (w/v), and about 0.01% to about 0.03% (w/v), e.g., about 0.02% (w/v).
  • the formulations of the invention may, optionally, further comprise a tonicity agent.
  • tonicity agents are used to adjust or maintain the osmolality of the formulations in order to bring it closer to the osmotic pressure of body fluids, such as blood or plasma.
  • Tonicity agents may also maintain the binding agent levels in a formulation.
  • the tonicity agent contributes to preserving the level, ratio, or proportion of the therapeutically active binding agent present in the formulation.
  • the term "tonicity” refers to the behavior of biologic components in a fluid enviornment or solution.
  • Isotonic solutions possess the same osmotic pressure as blood plasma, and can be intravenously infused into a subject without changing the osmotic pressure of the subject's blood plasma.
  • the tonicity agent is present in an amount sufficient to render the formulation suitable for intravenous infusion.
  • the tonicity agent serves as a bulking agent or a stabilizing agent as well.
  • the tonicity agent may allow the binding agent to overcome various stresses, such as freezing and shear.
  • Tonicity agents may include, but are not limited to, saccharides, sugars, glycerol, sorbitol, mannitol, sodium chloride, potassium chloride, magnesium chloride, and other inorganic salts. Those skilled in the art are aware that other tonicity agents can be used as long as they are pharmaceutically acceptable, i.e. suitable for administration to subjects.
  • the tonicity agent is present in the formulations in an amount from about 0.1% to 10% (w/v).
  • the tonicity agent may be present in the formulation in an amount of about 0.1% (w/v), about 0.2% (w/v), about 0.3% (w/v), about 0.4% (w/v), about 0.5% (w/v), about 0.6% (w/v), about 0.7% (w/v), about 0.8% (w/v), about 0.9% (w/v), about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 4.5% (w/v), about 5% (w/v), about 5.5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), and about 10% (w/v).
  • the tonicity agent may be present in the formulation in an amount from about 2% to about 8% (w/v), from about 3% to about 7% (w/v), and from about 4% to about 6% (w/v). In further alternative embodiments, the tonicity agent may be present in the formulation in an amount from about 0.1% to about 1%, from about 0.1% to about 0.5%, from about 0.1 to about 0.3%, and about 0.2%.
  • the tonicity agent is a saccharide.
  • saccharides include glucose, sucrose (which is also known as saccharose), maltose, trehalose, dextrose, xylitol, fructose and mannitol.
  • mannitol may be present in an amount of about 1% to about 10% (w/v), about 2% to about 8% (w/v), or about 3% to about 5% (w/v), e.g., about 4% (w/v).
  • sucrose which is also known as saccharose
  • sucrose may be present in an amount of about 1% to about 10% (w/v), about 3% to about 8% (w/v), or about 4% to about 6% (w/v), e.g., about 4.5, 5, 5.5, or 6% (w/v).
  • the tonicity agent is sodium chloride.
  • sodium chloride may be present in an amount of about 0.1% (w/v), about 0.2% (w/v), about 0.3% (w/v), about 0.4% (w/v), about 0.5% (w/v), about 0.6% (w/v), about 0.7% (w/v), about 0.8% (w/v), about 0.9% (w/v), and about 1% (w/v).
  • sodium chloride may be present in the formulation in an amount from about 0.1% to about 1%, from about 0.1% to about 0.5%, from about 0.1 to about 0.3%, and about 0.2%.
  • the formulations may comprise one or more tonicity agents.
  • the formulations may comprise one or more of the above tonicity agents in the above concentrations.
  • the formulations may comprise sucrose and sodium chloride, wherein each of the sucrose and sodium chloride concentrations is between about 0.1% to about 10% (w/v).
  • the sucrose concentration is about 6% and the sodium chloride concentration is about 0.2%.
  • the sucrose concentration is about 4.5% and the sodium chloride concentration is about 0.2%.
  • the osmolality of the formulations range from about 200 mOsm/kg to about 350 mOsm/kg, about 270 mOsm/kg to about 330 mOsm/kg, about 280 mOsm/kg to about 320 mOsm/kg, or about 290 mOsm/kg to about 310 mOsm/kg, e.g., about 300 mOsm/kg.
  • the formulations of the invention are, in some embodiments, substantially isotonic, i.e. having substantially the same osmotic pressure as human blood.
  • Osmolality can be measured by any means known to those of skill in the art, such as using vapor pressure or ice-freezing type osmometers.
  • the osmolality of the formulations of the invention can, for instance, be regulated by the one or more tonicity agents described herein.
  • compositions of the invention may, optionally, further comprise an amino acid.
  • amino acids include, but are not limited to, glycine, alanine, aspartic acid, lysine, serine, tyrosine, cysteine, glutamine, methionine, arginine, and proline.
  • the amino acid is present in the formulations in an amount from about 0.1% to 5% (w/v).
  • the amino acid may be present in the formulation in an amount of about 0.1% (w/v), about 0.2% (w/v), about 0.3% (w/v), about 0.4% (w/v), about 0.5% (w/v), about 0.6% (w/v), about 0.7% (w/v), about 0.8% (w/v), about 0.9% (w/v), about 1.0% (w/v), about 1.1% (w/v), about 1.2% (w/v), about 1.3% (w/v), about 1.4% (w/v), about 1.5% (w/v), about 1.6% (w/v), about 1.7% (w/v), about 1.8% (w/v), about 1.9% (w/v), about 2.0% (w/v), about 3% (w/v), about 4% (w/v), and about 5% (w/v).
  • the amino acid is present in the formulation in an amount from about 1.3% to about 1.8% (w/v), or about 1.4% to about 1.6% (w/v), e.g. , about 1.5% (w/v). In further alternative embodiments, the amino acid is present in the formulation in an amount from about 0.5% to about 1.5% (w/v), or about 0.8% to about 1.2% (w/v), e.g. , about 1.0% (w/v).
  • An exemplary amino acid is proline or arginine.
  • proline may be present in the formulation in an amount from about 1% to about 2%, (w/v) about 1.3% to about 1.8% (w/v), about 1.4% to about 1.6% (w/v), e.g., about 1.5% (w/v).
  • arginine may be present in the formulation in an amount from about 0.5% to about 1.5% (w/v), or about 0.8% to about 1.2% (w/v), e.g. , about 1.0% (w/v). vii.
  • Other excipients include 0.5% to about 1.5% (w/v), or about 0.8% to about 1.2% (w/v), e.g. , about 1.0% (w/v).
  • the formulations of the invention may comprise other excipients including, but not limited to, water for injection, diluents, solubilizing agents, soothing agents, additional buffers, inorganic or organic salts, antioxidants, or the like.
  • the formulations of the invention comprise no other excipients, except those described above.
  • Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed. (1980) may be included in the formulation provided that they do not adversely affect the desired characteristics of the formulation.
  • the formulation is substantially free of preservatives, although, in alternative embodiments, preservatives may be added as necessary.
  • cryoprotectants or lyoprotectants may be included in lyophilized formulations. viii. Liquid or lyophilized formulations
  • the formulations of the invention may either be liquid formulations or lyophilized formulations.
  • the formulations are liquid formulations.
  • the liquid formulations are ready for injection.
  • the formulations may be lyophilized powders.
  • the lyophilized powders are ready to be combined with a solvent just prior to administration. ix.
  • the invention provides a stable liquid antibody formulation suitable for subcutaneous administration, the formulation comprising: a) greater than about 80 mg/ml, e.g. , about 150 mg/ml, of a fully human IgG4 anti- LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8;
  • pH of the formulation is about pH 5.5
  • this formulation may be manufactured by:
  • a fully human IgG4 anti-LIGHT lymphotoxin-like, exhibits inducible expression and compete
  • step c) filtering the formulation from step b) under aseptic conditions using a sterilized, compatible membrane filter having a nominal pore size of 0.2 ⁇ , and then sterilizing the formulation by filtration under aseptic conditions into sterilized containers made out of inert material using a sterilized, compatible membrane filter having a nominal pore size of 0.2 ⁇ ; d) filling the formulation from step c) under aseptic conditions into sterilized vials that are closed with stoppers and flip-off caps with a flange; and, optionally,
  • step e) inspecting the containers from step d) for coarse contaminants, intact sealing, and visible particles.
  • the invention provides a stable liquid antibody formulation suitable for intravenous administration, the formulation comprising:
  • pH of the formulation is about pH 5.5.
  • the invention provides a stable lyophilized antibody formulation suitable for intravenous administration, the formulation comprising:
  • pH of the formulation is about pH 5.5.
  • the invention provides a stable antibody formulation comprising: a) about 20 mg/mL of a humanized IgG4 anti-CXCR5 (C-X-C chemokine receptor type 5) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;
  • pH of the formulation is about pH 6.0.
  • the invention provides a stable antibody formulation comprising:
  • a humanized IgG4 anti-CXCR5 (C-X-C chemokine receptor type 5) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;
  • the formulations of the invention are stable at 5°C for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or more, and typically at least about 12, 18 or 24 months or more. In exemplary embodiments, they are stable at 5°C for at least about 6 months or more. In other exemplary embodiments, they are stable at 5°C for at least about 9 months. In further exemplary embodiments, they are stable at 5°C for at least about 1 year or more, and typically about 2 years.
  • the formulations are suitable for administration parenterally, intravenously, intramuscularly, intradermally, subcutaneously, or a combination thereof.
  • the formulations of the invention are suitable for delivery by a variety of techniques.
  • the formulation is administered intravenously.
  • the formulations are typically sterile.
  • Methods for making formulations sterile are well known in the art and include, for example, filtration through sterile filtration membranes or autoclaving the ingredients of the formulation, with the exception of the antibodies, at about 120°C for about 30 minutes.
  • the invention provides a stable liquid antibody formulation suitable for intravenous administration, the formulation comprising: a) about 5 to about 80 mg/mL, e.g.
  • the invention provides a stable antibody formulation comprising:
  • the formulation is administered subcutaneously.
  • a stable liquid antibody formulation comprising: a) about 150 mg/mL of a fully human IgG4 anti-LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8; b) about 10 mM citrate buffer; c) about 0.005% (w/v) polysorbate 20; d) about 4% (w/v) mannitol; and wherein the pH of the formulation is about pH 5.5.
  • the invention provides a stable antibody formulation comprising: a) about 100 mg/mL of a humanized IgG4 anti-CXCR5 (C-X- C chemokine receptor type 5) antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26; b) about 10 mM citrate buffer; c) about 0.01% polysorbate 20; d) about 4.5% sucrose; e) about 0.2% sodium chloride; and f) about 1% arginine; wherein the pH of the formulation is about pH 6.0.
  • Effective doses of the formulations of the invention vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the subject is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages may need to be titrated to optimize safety and efficacy.
  • the formulations of the invention may be administered on multiple occasions. Intervals between single dosages can be daily, weekly, biweekly, monthly or yearly. Intervals can also be irregular. In some methods, the dosage is adjusted to achieve a certain plasma binding agent, such as an antibody, concentration. Dosage and frequency will vary depending on the half-life of the anti-LIGHT or anti-CXCR5 binding agent, such as an antibody, in the subject. In general, human antibodies show the longest half- life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies.
  • the invention provides a pharmaceutical unit dosage form comprising a therapeutically effective amount of a formulation of the invention for the treatment of one or more diseases in a subject through administration of the dosage form to the subject.
  • the subject is a human.
  • the human may be an adult or may be an infant.
  • pharmaceutical unit dosage form refers to a physically discrete unit suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic/prophylactic effect in association with the required citrate buffer and pH.
  • the unit dosage form may be a container comprising the formulation. Suitable containers include, but are not limited to, sealed ampoules, vials, bottles, syringes, and test tubes.
  • the containers may be formed from a variety of materials, such as glass or plastic, and may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container is a vial. Generally, the container should maintain the sterility and stability of the formulation.
  • the formulations are packaged in 2 mL vials that are made of clear, colorless type I glass, and closed with a stopper (fluoropolymer-coated bromobutyl) sealed with flip-of caps with flange (polypropylene).
  • the vials are, in some embodiments, filled with 1.2 mL of the formulations so that the vial has an overfill volume of about 0.2 mL per vial, and an extractable volume of 1.0 mL.
  • the formulations are secondarily packaged in a container, such as a cardboard box, that protects the vials from light.
  • the methods comprising administering a formulation of the invention to a subject.
  • the invention further relates to a formulation of the invention for use in a herein- described method for treating a LIGHT-mediated disease or disorder.
  • the LIGHT-mediated disease is a chronic bowel disease, or an inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC).
  • IBD inflammatory bowel disease
  • CD Crohn's disease
  • UC ulcerative colitis
  • the LIGHT mediated disease is graft- versus- host disease (GVHD).
  • the anti-CXCR5 binding agent is used for reduction of signs and symptoms, inhibition of progression of structural damage, induction of a major clinical response, and prevention of disability in adult patients with moderately to severely active Rheumatoid Arthritis (RA) who have had inadequate response to one or more Disease-Modifying Anti-Rheumatic Drugs (DMARDs), such as methotrexate (MTX), or TNFa antagonists.
  • DMARDs Disease-Modifying Anti-Rheumatic Drugs
  • MTX methotrexate
  • TNFa antagonists such as methotrexate (MTX)
  • the anti-CXCR5 binding agent may be used in combination with DMARDs or anti-TNFa agonists.
  • the formulations of the invention can be administered in combination with one or more therapies (e.g. , therapies that are not the formulations of the invention that are currently administered to prevent, treat, manage, and/or ameliorate a LIGHT- mediated disease or a CXCR5- mediated disease.
  • therapies e.g. , therapies that are not the formulations of the invention that are currently administered to prevent, treat, manage, and/or ameliorate a LIGHT- mediated disease or a CXCR5- mediated disease.
  • a first therapy can be administered before (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), concurrently, or after (e.g.
  • any additional therapy can be administered in any order with the other additional therapies.
  • therapies that can be administered in combination with an antibody of the invention include approved antiinflammatory agents listed in the U.S. Pharmacopoeia and/or Physician's Desk Reference.
  • kits comprising a formulation of the invention.
  • the kit may further comprise one or more containers comprising pharmaceutically acceptable excipients, and include other materials desirable from a commercial and user standpoint, including filters, needles and syringes.
  • Associated with the kits can be instructions customarily included in commercial packages of therapeutic, prophylactic or diagnostic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contra-indications, and/or warnings concerning the use of such therapeutic, prophylactic or diagnostic products.
  • the kit can also be associated with a label that can be any kind of data carrier (e.g. , a leaflet, sticker, chip, print or bar code) comprising information.
  • the kit can further comprise a device for administration of the formulation, and particularly a device that contains the formulation, i.e. , a pre-filled device such as, but not limited to, a pre- filled syringe or a pre-filled autoinjector.
  • a pre-filled device such as, but not limited to, a pre- filled syringe or a pre-filled autoinjector.
  • the kit can also comprise a container comprising the formulation, i.e., a pre-filled container, such as a pre-filled vial, cartouche, sachet, or ampoule.
  • any of the herein described embodiments can be combined with one or more of the other herein described embodiments unless explicitly stated to the contrary.
  • any of the herein described binding agents and antibodies and the herein described formulations thereof can be used in combination with any of the kits, pre-filled devices or pre-filled containers, or can be used in the methods of treatment or medical uses as described herein in connection with the respective antibody ⁇ e.g., the stable formulations comprising the anti-LIGHT antibodies or anti-CXCR5 antibodies can be combined with any of the herein described kits, containers or devices).
  • binding agents specifically binding an antigen ⁇ e.g., a binding agent specifically binding LIGHT or a binding agent specifically binding CXCR5
  • a binding agent specifically binding LIGHT or a binding agent specifically binding CXCR5 can also be used in any of the methods of treatment that are described herein in connection with the respective antibodies ⁇ i.e., anti-LIGHT or anti-CXCR5) and vice versa.
  • a fully human IgG4 anti-LIGHT antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8 (the "Lead LIGHT Antibody”) was used in Examples 1-9 in order to determine optimal formulation conditions.
  • the Lead Antibody formulated in phosphate buffered saline (PBS) at a concentration of 5.5 mg/mL and at a pH of 7.3 (the "Original Formulation”, “PBS Formulation”, or “Reference Lot”), was used in the following examples.
  • PBS phosphate buffered saline
  • Table 1 shows the excipients that were used in the following examples, which were chosen according to their acceptability/suitability for use in parenteral products.
  • All buffers were manufactured under stirring to dissolve the respective excipients. pH was adjusted using 0.1 M HCl or 0.1 M NaOH. The general concentration of all buffers was 10 mM.
  • the protein concentrations of all antibody solutions were measured against buffer using a NanoDrop NDIOOO. Proteins concentrations near or below 5 mg/mL were diluted 1:3, while higher protein concentrations near 20 mg/mL were diluted 1:20, and the absorption was measured at 215 nm and 280 nm.
  • the hydrodynamic diameter of the molecule was measured using laser light scattering.
  • the samples were sterile filtered prior to the analytics if turbidity was observed, thus only soluble aggregates could be detected.
  • DSC Differencial scanning calorimetry
  • Density of the formulations was measured using a falling sphere viscosimeter DMA4500 Anton Paar.
  • SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • Antigen-ELISA Antigen-Enzyme Linked Immunosorbent Assay
  • Antigen-ELISA was performed to determine the functionality of the antibody. The binding properties to native LIGHT protein were monitored in comparison to the current standard of the antibody. This potency was reported as the relative EC 50 . Isoelectric focusing (IEF)
  • IEF IEF was performed.
  • the isoelectric pattern was specific for the Lead Antibody and served as an identification test. Degradation could be seen by a different charge pattern.
  • Table 2 shows a summary of all of the formulations that were prepared and analyzed in the following examples. Each of the formulations contained the Lead LIGHT Antibody at the concentration listed.
  • Formulation 11 Sucrose 5 % 50 mg/mL lyo
  • Formulation 12 Citrate 10 mM 7.0 5 mg/mL
  • the Reference Lot was characterized. As stated in the Materials section above, the Reference Lot contains the Lead LIGHT Antibody formulated in phosphate buffered saline (PBS) at a concentration of 5.5 mg/mL and at a pH of 7.3, and produced in research solutions Vitry (BioSCP).
  • PBS phosphate buffered saline
  • Isoelectric focusing was used to determine the isoelectric point (pi) of the Lead Antibody.
  • the pi of the Lead LIGHT Antibody was theoretically calculated as 6.28, and then measured by denaturated isoelectric focusing using standard methods known in the art. As shown in Figure 1, the main bands show that the pi of the Lead LIGHT Antibody was 6.8-7.2.
  • FIG. 1 shows an SDS-PAGE gel that compared different Reference Lot batches under reducing and non-reducing conditions.
  • An ELISA was used to determine the antigen binding activity of the Lead LIGHT Antibody.
  • Figure 3 shows an ELISA graph that was used to determine the antigen binding activity of the first and second batches of Reference Lot.
  • SEC was used to determine the presence of aggregates, as well as degradation products of the first batch of Reference Lot.
  • size exclusion chromatography detected high molecular weight proteins (HMWP), e.g., di-/oligomers (RRT0.8) or aggregates, and low molecular weight proteins (LMWPs) or degradation products.
  • HMWP high molecular weight proteins
  • RRT0.8 di-/oligomers
  • LMWPs low molecular weight proteins
  • the first batch of Reference Lot had a purity of 97% monomer content.
  • WCX was used to monitor the charge heterogeneity of the first batch of Reference Lot.
  • rearrangements of acidic, neutral, and basic isoforms occured during stability studies.
  • the first batch of Reference Lot had a distribution of acidic/neutral/basic isoforms of 42.3/55.6/1.9%.
  • DSC was used to analyze the unfolding temperature Tm of the first batch of Reference Lot. As shown in Figure 6, the three domains of the antibody unfold at 68°C, 75°C, and 78°C.
  • DLS was used to determine the hydrodynamic diameter of the antibody monomer and potential soluble aggregates. As shown in Figures 7 & 8, a hydrodynamic diameter of about 10 nm was detected, but aggregates were seen in PBS. However, aggregates were not seen in citrate buffer (Figure 10).
  • the Original Formulation exhibited aggregates; half-molecules; degradation products; low molecular weight proteins (LMWPs); high molecular weight proteins (HMWPs); and rearrangements of acidic, basic, and neutral antibody isoforms (see Example 1).
  • LMWPs low molecular weight proteins
  • HMWPs high molecular weight proteins
  • rearrangements of acidic, basic, and neutral antibody isoforms see Example 1.
  • Formulations of the Lead LIGHT Antibody (a fully human IgG4 anti-LIGHT antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8) containing lOmM citrate buffer at a pH of 5, 5.5, and 6, with and without polysorbate 20 were tested.
  • Table 3 shows the analytical results of the first batch of Reference Lot, and the various experimental formulations of the Lead LIGHT Antibody formulated into citrate, at a pH of 5.0 and 5.5 and 6.0, with and without polysorbate 20. Aggregates were found in dynamic light scattering (DLS) measurements for the Reference Lot, but not in all other tested formulations.
  • DLS dynamic light scattering
  • Tm as measured by differencial scanning calorimetry ⁇ DSC
  • SEC size exclusion chromatography
  • Table 6 shows the analytical results of the first batch of high concentration (about 40 mg/ml) antibody formulations: high phosphate buffered saline (PBS) at a pH of 7.3 (Formulation 2) or citrate at a pH of 5.5 with polysorbate 20 (Formulation 4).
  • PBS high phosphate buffered saline
  • Formulation 4 citrate at a pH of 5.5 with polysorbate 20
  • Table 9 shows the freeze drying program that was used in this example.
  • Table 10 shows the analytical results of the first batch of Reference Lot, and the various experimental lyophilized formulations of the Lead LIGHT Antibody formulated into various combinations of citrate buffer, sucrose, polysorbate 20, and proline.
  • HMWPs high molecular weight proteins
  • LMWPs low molecular weight proteins
  • Table 13 shows the analytical results of the first batch of Reference Lot, and the various xperimental formulations of the Lead LIGHT Antibody formulated into various combinations of citrate buffer or histidine buffer.
  • the citrate formulation of the invention appeared in all experiments to perform better than histidine.
  • citrate formulations had a higher monomer content compared to the both the Reference Lot batch and the histidine (Table 13) and the content or low molecular weight proteins (LMWPs) and high molecular weight proteins (HMWPs) were also significantly lower (Table 14). As before, these differences could not be detected with SDS- PAGE analysis (Table 15).
  • Formulation development was performed on the Lead LIGHT Antibody with the goal of developing a liquid dosage form with an acceptable shelf life when stored at +2 to +8°C.
  • Preliminary stress studies showed the formation of subvisible and visible particles, high molecular weight species and more basic species. Therefore, these parameters were monitored during the screening of formulation candidates using visual assessment, dynamic light scattering, light obscuration, size exclusion chromatography, sodium dodocyl sulphate polyacrylamide gel electrophoresis, and weak cationic exchange chromatography.
  • Different liquid formulations were used in the pre- formulation and formulation trials prior to selection of the clinical formulation. According to the findings, a formulation in 10 mM citrate buffer adjusted to pH 5.5 (Formulation 14) was selected for further development.
  • the pH of the formulation is in the region of optimal physical and chemical stability of the drug substance and acceptable physiological tolerability (e.g. , osmolarity).
  • Formulation 14 is a solution for injection and is an aqueous, sterile, and clear solution containing the Lead LIGHT Antibody, sodium citrate dihydrate (buffering agent), polysorbate 20 (stabilizing agent), and mannitol (tonicity agent).
  • Sodium hydroxide solution and hydrochloric acid were used to adjust the pH to 5.5.
  • a GMP-compliant manufacturing process was developed for the subcutaneous, high- concentration antibody formulation (Formulation 14) of Example 6.
  • the manufacturing procedure consisted of dissolving, pH adjustment, sterile filtration, filling, and packaging steps.
  • Drug substance (the Lead LIGHT Antibody) is provided in a liquid form in the formulation buffer (10 mM citrate buffer at pH 5.5).
  • the excipients were all water-soluble and dissolved in the initial aqueous portion of the formulation buffer during manufacture.
  • the bulk drug substance solution was further diluted with the same formulation buffer to reach the concentration of 150 mg/mL of Lead LIGHT Antibody.
  • the bulk solution was well mixed to facilitate the dissolution process and to ensure homogeneity.
  • Sterilization by filtration was carried out (according to Ph. Eur. and USP) using bacteria retentive filters having a nominal pore size of 0.2 ⁇ . An additional filtration procedure before "sterilization by filtration” was performed to ensure a low bioburden.
  • Bioburden sampling was done before the pre-filtration step as well as the sterile filtration step.
  • Vials were filled to about 1.2 mL to ensure an extractable volume of 1.0 mL.
  • the 2 mL vials were made of clear, colorless type I glass, and closed with a stopper (fluoropolymer- coated bromobutyl) sealed with flip-off caps with a flange (polypropylene).
  • the primary packaging materials met the requirements of the Ph. Eur. and USP. The suitability of the primary packaging materials was substantiated by the results of the stability tests.
  • the vials were filled with a volume of 1.2 mL to ensure an extractable volume of 1.0 mL.
  • Sodium citrate dihydrate was dissolved in water for injection while stirring in a vessel made of inert material (e.g. , stainless steel or glass), until completely dissolved.
  • the pH value was adjusted to 5.5 using hydrochloric acid, diluted (e.g. , 0.1 M hydrochloric acid) and/or sodium hydroxide solution (e.g. , 0.1 M sodium hydroxide), if necessary.
  • Lead Antibody, mannitol, and polysorbate 20 were diluted in the buffer solution from step 1 while stirring in a vessel made of inert material (e.g. , stainless steel or glass) until completely dissolved. If necessary, the pH value was adjusted to 5.5 using hydrochloric acid, diluted (e.g. , 1 M hydrochloric acid) or sodium hydroxide solution (e.g. , 1 M sodium hydroxide). Buffer solution from step 1 (remainder) was added to adjust the final weight.
  • inert material e.g. , stainless steel or glass
  • Solution from step II was filtered under aseptic conditions using a sterilized, compatible membrane filter (e.g. , polyether sulfone or polyvinylidene difluoride) having a nominal pore size of 0.2 ⁇ .
  • a sterilized, compatible membrane filter e.g. , polyether sulfone or polyvinylidene difluoride having a nominal pore size of 0.2 ⁇ .
  • Solution from step III. a was sterilized by filtration under aseptic conditions into sterilized containers made out of inert material (e.g. , stainless steel or glass) using a sterilized, compatible membrane filter (e.g. , polyether sulfone or polyvinylidene difluoride) having a nominal pore size of 0.2 ⁇ .
  • inert material e.g. , stainless steel or glass
  • a sterilized, compatible membrane filter e.g. , polyether sulfone or polyvinylidene difluoride having a nominal pore size of 0.2 ⁇ .
  • step Ill.b Solution from step Ill.b was filled under aseptic conditions into sterilized vials, which were closed with stoppers and flip-off caps with a flange.
  • step IV The containers from step IV were inspected for coarse contaminants, intact sealing, and visible particles.
  • containers e.g. , cardboard boxes.
  • DLS was used to determine the hydrodynamic diameter of the antibody monomer and potential soluble aggregates. As shown in Figure 10, aggregates were not seen in citrate buffer. However, as shown in Figures 7 & 8, aggregates were seen in PBS. Due to the higher concentration of antibody, an increase in ZAve to 28 nm was observed, compared to the sample in PBS.
  • Drug product Lead LIGHT Antibody Batch no.: 11 021
  • Container/closure 2 mL glass vials
  • Drug product Lead LIGHT Antibody Batch no.: 11 021 solution for injection
  • Container/closure 2 mL glass vials
  • Drug product Lead LIGHT Batch no.: 11_021
  • Container/closure 2 mL glass vials
  • Lead LIGHT Antibody_11_30B 212 1.059 22.58 39 1.3 98.7 0.0
  • Lead LIGHT Antibody _11_30B 5°C 212 1.3 98.7 0.0
  • a humanized IgG4 anti-CXCR5 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26 (the "Lead CXCR5 Antibody") was used in Examples 10-12 in order to determine optimal formulation conditions for a 20 mg/mL formulation.
  • the Lead Antibody is a humanized monoclonal antibody (mAB) specific to human CXCR5, with an engineered IgG4 framework containing 2 amino acid substitutions aimed at reducing half-molecules (S241P) and effector functions (L248E).
  • the Lead CXCR5 Antibody protein structure is comprised of two kappa light chains, each with a molecular weight of approximately 24 kDa, and two IgG4 heavy chains, each with a molecular weight of approximately 48 kDa linked through disulfide bridges. Each light chain consists of 219 amino acid residues, and each heavy chain consists of 437 amino acid residues.
  • Examples 10-12 were collected during preformulation activities for the Lead CXCR5 Antibody and its drug product for intravenous and subcutaneous administration.
  • the objective of the preformulation studies was to provide good stability of buffered Lead CXCR5 Antibody solutions with a target concentration of 20 mg/mL, with special emphasis on the aggregation behavior of the Lead CXCR5 Antibody and its tendency to form half- molecules, as the Lead Antibody is an IgG4 subclass antibody, which is prone to aggregation and the formation of particles.
  • the Lead CXCR5 Antibody batch RSN0151 was formulated in PBS pH 7.2 with a concentration of 5.13 mg/mL. Excipients
  • Table 23 shows excipients that were used during the preformulation studies.
  • the equipment was placed inside a clean-bench under aseptic conditions and the process was performed at room temperature.

Abstract

La présente invention concerne des formulations stables d'anticorps pharmaceutiques, comprenant des formulations de produit de médicament liquides et des formulations de produit de médicament lyophilisées, comprenant un agent de liaison à IgG4 et un tampon citrate, le pH de la formulation étant inférieur ou égal à la fois à pH 6 et au pi de l'agent de liaison. Les formulations peuvent être utilisées dans le traitement de maladies intestinales chroniques ou de la polyarthrite rhumatoïde.
PCT/US2013/033881 2012-03-26 2013-03-26 Formulations stables d'agent de liaison à igg4 WO2013148686A2 (fr)

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ES13714505T ES2702246T3 (es) 2012-03-26 2013-03-26 Formulaciones de agente de unión IgG4 estables
EP13714505.8A EP2830658B1 (fr) 2012-03-26 2013-03-26 Formulations stables d'agent de liaison à igg4
JP2015503464A JP6265970B2 (ja) 2012-03-26 2013-03-26 安定なＩｇＧ４に基づく結合剤の製剤
EP18190556.3A EP3431104A1 (fr) 2012-03-26 2013-03-26 Formulations d'agents de liaison igg4 stables
BR122019026701-4A BR122019026701B1 (pt) 2012-03-26 2013-03-26 Formulações de agentes de ligação à base de igg4 estáveis, kit, e dispositivo ou recipiente pré-cheios
BR112014023952-5A BR112014023952B1 (pt) 2012-03-26 2013-03-26 Formulações de agentes de ligação à base de igg4 estáveis, kit, e dispositivo ou recipiente pré-cheios
CA2868401A CA2868401C (fr) 2012-03-26 2013-03-26 Preparations d'anticorps igg4 stables anti-cxcr5
RU2014142990A RU2644214C2 (ru) 2012-03-26 2013-03-26 СТАБИЛЬНЫЕ ПРЕПАРАТЫ СВЯЗЫВАЮЩЕГО СРЕДСТВА НА ОСНОВЕ IgG4
PL13714505T PL2830658T3 (pl) 2012-03-26 2013-03-26 Stabilne formulacje środków wiążących lgG4
HK15105917.2A HK1204990A1 (en) 2012-03-26 2015-06-22 Stable igg4 binding agent formulations igg4

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CA3123252A1 (fr) 2013-10-03
EP2830658B1 (fr) 2018-10-10
PL2830658T3 (pl) 2019-02-28
JP2015514090A (ja) 2015-05-18
EP2830658A2 (fr) 2015-02-04
EP3431104A1 (fr) 2019-01-23
RU2644214C2 (ru) 2018-02-08
JP6265970B2 (ja) 2018-01-24
BR112014023952B1 (pt) 2023-01-24
CA3123252C (fr) 2023-08-22
RU2014142990A (ru) 2016-05-20
CA2868401A1 (fr) 2013-10-03
BR122019026701B1 (pt) 2023-01-24
CA3204402A1 (fr) 2013-10-03
CA2868401C (fr) 2021-08-24
BR112014023952A2 (pt) 2017-07-18
WO2013148686A3 (fr) 2013-11-28

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